Catalysts for Nitrogen Fixation: Nitrogenases, Relevant Chemical Models and Commercial Processes (Nitrogen Fixation: Origins, Applications, and Research Progress, 1) 1402025084, 9781402025082

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Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment

Nitrogen Fixation: Origins, Applications, and Research Progress VOLUME 4

Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment Edited by

Dietrich Werner Philipps-University, Marburg, Germany and

William E. Newton Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, U.S.A.

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN-10 1-4020-3542-X (HB) ISBN-10 1-4020-3544-6 (e-book) ISBN-13 978-1-4020-3542-9 (HB) ISBN-13 978-1-4020-3544-5 (e-book)

Published by Springer, P.O. Box 17, 3300 AA Dordrecht, The Netherlands. www.springeronline.com Background figure caption: “A seed crop of clover (Trifolium hirtum) in flower near Moora, Western Australia. Photograph courtesy of Mike Davies, Senior Technical Officer, Pasture Research Group of Agriculture WA and reproduced with permission.” Vol. 4-specific figure caption: “First year soybean cultivation from a field trial in Puerto Rico with both inoculated and non-inoculated rows. Photograph courtesy of R. Stewart Smith, Nitragin Company, USA, and reproduced with permission.”

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TABLE OF CONTENTS Series Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii List of Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii Chapter 1. Production and Biological Nitrogen Fixation of Tropical Legumes D. Werner . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2. Phaseolus sp. and Vigna sp. (Beans) . . . . . . . . . . . . . . . . . . . . . . . . . 2 3. Arachis hypogaea (Groundnut, Peanut) . . . . . . . . . . . . . . . . . . . . . . . 7 4. Cicer arietinum (Chickpea) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5. Cajanus cajan (Pigeon pea) . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 9 6. Mucuna pruriens (Velvet bean) and Other Legumes . . . . . . . . . . . . 10 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Chapter 2. Nitrogen Fixation by Soybean in North America S. G. Pueppke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Soybean: Pathways to North America and Establishment as a Crop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Soybean Production in North America . . . . . . . . . . . . . . . . . . . . . . . 3. Major Soybean Cropping Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Biological Nitrogen Fixation by Soybean in North America . . . . . 5. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 15 17 20 20 21 22 22

Chapter 3. The Importance of Nitrogen Fixation to Soybean Cropping in South America M. Hungria, J. C. Franchini, R. J. Campo and P. H. Graham. . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Taxonomy, Origins, and Importance of Soybean . . . . . . . . . . . . . . 3. Biological Nitrogen Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Economic Importance of Biological Nitrogen Fixation (BNF) in South America . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Crop Management in South America . . . . . . . . . . . . . . . . . . . . . . . . 6. Final Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

32 34 38 39 39

Chapter 4. Production, Regional Distribution of Cultivars, and Agricultural Aspects of Soybean in India S. K. Mahna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction and Historical Background . . . . . . . . . . . . . . . . . . . . .

43 43

v

25 25 25 29

vi 2. All-India Area Coverage, Productivity, and Production of Soybean between 1970-2003 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. All-India State-wise Area Coverage, Productivity, and Production of Soybean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Regional Distribution of Soybean Cultivars . . . . . . . . . . . . . . . . . . 5. Regional Agricultural Aspects of Soybean Cultivation . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 5. Soybean Cultivation and BNF in China J. E. Ruiz Sainz, J. C. Zhou, D.-N. Rodriguez-Navarro, J. M. Vinardell and J. E. Thomas-Oates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Soybean Cultivation in China: Historical Aspects and Current Situation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Nitrogen-Fixing Bacteria that Nodulate Soybean . . . . . . . . . . . . . . 4. The Soybean Germplasm Collection in China . . . . . . . . . . . . . . . . 5. Soybean in Crop Rotation and in Continuous Cultivation . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45 46 49 59 64 64

67 67 68 75 81 82 84 85 85

Chapter 6. Soil Stress Factors Influencing Symbiotic Nitrogen Fixation M. J. Sadowsky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 2. Importance of Symbiotic Nitrogen Fixation . . . . . . . . . . . . . . . . . . . 89 3. Symbiotic Interaction of Legumes with Rhizobia . . . . . . . . . . . . . . 90 4. Nodulation and Nitrogen-Fixation Genetics in the Rhizobia and Bradyrhizobia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 5. Rhizobia in the Soil Environment . . . . . . . . . . . . . . . . . . . . . . . . . . 94 6. Stress Factors in the Soil Environment that Influence N2 Fixation . . 95 7. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Chapter 7. Nodulated Legume Trees J. I. Sprent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 2. Leguminosae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 3. Rhizobia that Nodulate Legume Trees . . . . . . . . . . . . . . . . . . . . . . 133 4. Types of Nodule formed on Trees . . . . . . . . . . . . . . . . . . . . . . . . . . 134 5. Mycorrhizas and Other Nutrient-Acquisition Systems . . . . . . . . . . 135 6. Measurement of Nitrogen Fixation by Trees . . . . . . . . . . . . . . . . . . 136 7. Role of Legume Trees in Natural and Managed Systems . . . . . . . . 137 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

vii Chapter 8. Nitrogen Fixing Trees with Actinorhiza in Forestry and Agroforestry R. O. Russo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 2. General Characteristics of the Actinorhizal Symbiosis . . . . . . . . . . 144 3. Host Botanical Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 4. Nitrogen Fixation in Actinorhizal Trees . . . . . . . . . . . . . . . . . . . . . . 149 5. Mycorrhizal Associations with Actinorhizal Trees . . . . . . . . . . . . . 153 6. Actinorhizal Trees in Agroforestry . . . . . . . . . . . . . . . . . . . . . . . . . 157 7. The Genus Casuarina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 8. The Experience of the Central America Fuelwood Project . . . . . . . 159 9. The Case of Alnus acuminata in Tropical Highlands . . . . . . . . . . . 161 10. Other Uses of Actinorhizal Trees . . . . . . . . . . . . . . . . . . . . . . . . . 163 11. Concluding Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Chapter 9. Molecular Ecology of N2-fixing Microbes Associated with Gramineous Plants: Hidden Activities of Unknown Bacteria T. Hurek and B. Reinhold-Hurek . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Problem of Identifying Key Diazotrophic Bacteria in Gramineous Plants: The Classical Approach . . . . . . . . . . . . . . 3. The Significance of Diazotrophic Grass Endophytes . . . . . . . . . . . 4. The Significance of Culture-Independent Methods . . . . . . . . . . . . . 5. The Significance of nifH-Targeted Methods . . . . . . . . . . . . . . . . . . 6. Limitations of nifH-Targeted Methods . . . . . . . . . . . . . . . . . . . . . . 7. Many Diazotrophs Defy Cultivation . . . . . . . . . . . . . . . . . . . . . . . . 8. Diazotrophic Grass Endophytes as Key Organisms for BNF in Gramineous Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

173 173 174 176 179 180 181 183 185 189 191

Chapter 10. Interactions of Arbuscular Mycorrhiza and Nitrogen-Fixing Symbiosis in Sustainable Agriculture J. M. Barea, D. Werner, C. Azcón-Guilar and R. Azcón . . . . . . . . . . . . . . . . . . 199 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 2. Purpose of Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 3. Nitrogen-Fixing Symbioses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 4. Arbuscular Mycorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 5. Interactions between AM Fungi and Rhizobia to Improve Legume Productivity in Agriculture . . . . . . . . . . . . . . . . . . . . . . . 205 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

viii Chapter 11. Inoculant Preparation, Production and Application M. Hungria, M. F. Loureiro, I. C. Mendes, R. J. Campo and P. H. Graham . . . 223 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 2. Strain Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 3. Inoculant Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 4. Inoculant Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 5. Factors Affecting the Success of Inoculation . . . . . . . . . . . . . . . . . 240 6. Main Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 Chapter 12. Nitrifying Bacteria C. Fiencke, E. Spieck and E. Bock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 1. Nitrification as Part of the Nitrogen Cycle . . . . . . . . . . . . . . . . . . . 255 2. Two Physiological Groups of Bacteria Contribute to Nitrification . .257 3. Ecology and Detection of Nitrifying Bacteria . . . . . . . . . . . . . . . . . 258 4. Metabolism of Nitrifying Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 260 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Chapter 13. The Nitrogen Cycle: Denitrification and its Relationship to N2 Fixation R. J. M. van Spanning, M. J. Delgado and D. J. Richardson . . . . . . . . . . . . . . . 277 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 2. The Nitrogen Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 3. Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 4. Bacterial Respiratory Nitrate Reductases . . . . . . . . . . . . . . . . . . . . . 282 5. Nitrite Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 6. Nitric Oxide Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 7. Nitrous Oxide Reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 8. Linkage of the Denitrification Gene Clusters . . . . . . . . . . . . . . . . . . 307 9. Bioenergetics of Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310 10. Regulation of Transcription of Denitrification Genes . . . . . . . . . . 312 11. Regulation of Denitrification by Environmental Factors . . . . . . . 320 12. Diversity of Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 13. Yeast and Fungal Denitrification . . . . . . . . . . . . . . . . . . . . . . . . . . 325 14. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

SERIES PREFACE Nitrogen Fixation: Origins, Applications, and Research Progress Nitrogen fixation, along with photosynthesis as the energy supplier, is the basis of all life on Earth (and maybe elsewhere too!). Nitrogen fixation provides the basic component, fixed nitrogen as ammonia, of two major groups of macromolecules, namely nucleic acids and proteins. Fixed nitrogen is required for the N-containing heterocycles (or bases) that constitute the essential coding entities of deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), which are responsible for the high-fidelity storage and transfer of genetic information, respectively. It is also required for the amino-acid residues of the proteins, which are encoded by the DNA and that actually do the work in living cells. At the turn of the millennium, it seemed to me that now was as good a time as any (and maybe better than most) to look back, particularly over the last 100 years or so, and ponder just what had been achieved. What is the state of our knowledge of nitrogen fixation, both biological and abiological? How has this knowledge been used and what are its impacts on humanity? In an attempt to answer these questions and to capture the essence of our current knowledge, I devised a seven-volume series, which was designed to cover all aspects of nitrogen-fixation research. I then approached my long-time contact at Kluwer Academic Publishers, Ad Plaizier, with the idea. I had worked with Ad for many years on the publication of the Proceedings of most of the International Congresses on Nitrogen Fixation. My personal belief is that congresses, symposia, and workshops must not be closed shops and that those of us unable to attend should have access to the material presented. My solution is to capture the material in print in the form of proceedings. So it was quite natural for me to turn to the printed word for this detailed review of nitrogen fixation. Ad’s immediate affirmation of the project encouraged me to share my initial design with many of my current co-editors and, with their assistance, to develop the detailed contents of each of the seven volumes and to enlist prospective authors for each chapter. There are many ways in which the subject matter could be divided. Our decision was to break it down as follows: nitrogenases, commercial processes, and relevant chemical models; genetics and regulation; genomes and genomics; associative, endophytic, and cyanobacterial systems; actinorhizal associations; leguminous symbioses; and agriculture, forestry, ecology, and the environment. I feel very fortunate to have been able to recruit some outstanding researchers as coeditors for this project. My co-editors were Mike Dilworth, Claudine Elmerich, John Gallon, Euan James, Werner Klipp, Bernd Masepohl, Rafael Palacios, Katharina Pawlowski, Ray Richards, Barry Smith, Janet Sprent, and Dietrich Werner. They worked very hard and ably and were most willing to keep the volumes moving along reasonably close to our initial timetable. All have been a pleasure to work with and I thank them all for their support and unflagging interest.

ix

x Nitrogen-fixation research and its application to agriculture have been ongoing for many centuries – from even before it was recognized as nitrogen fixation. The Romans developed the crop-rotation system over 2000 years ago for maintaining and improving soil fertility with nitrogen-fixing legumes as an integral component. Even though crop rotation and the use of legumes was practiced widely but intermittently since then, it wasn’t until 1800 years later that insight came as to how legumes produced their beneficial effect. Now, we know that bacteria are harbored within nodules on the legumes’ roots and that they are responsible for fixing N2 and providing these plants with much of the fixed nitrogen required for healthy growth. Because some of the fixed nitrogen remains in the unharvested parts of the crop, its release to the soil by mineralization of the residue explains the follow-up beneficial impact of legumes. With this realization, and over the next 100 years or so, commercial inoculants, which ensured successful bacterial nodulation of legume crops, became available. Then, in the early 1900’s, abiological sources of fixed nitrogen were developed, most notable of these was the Haber-Bosch process. Because fixed nitrogen is almost always the limiting nutrient in agriculture, the resulting massive increase in synthetic fixed-nitrogen available for fertilizer has enabled the enormous increase in food production over the second half of the 20th century, particularly when coupled with the new “green revolution” crop varieties. Never before in human history has the global population enjoyed such a substantial supply of food. Unfortunately, this bright shiny coin has a slightly tarnished side! The abundance of nitrogen fertilizer has removed the necessity to plant forage legumes and to return animal manures to fields to replenish their fertility. The result is a continuing loss of soil organic matter, which decreases the soil’s tilth, its waterholding capacity, and its ability to support microbial populations. Nowadays, farms do not operate as self-contained recycling units for crop nutrients; fertilizers are trucked in and meat and food crops are trucked out. And if it’s not recycled, how do we dispose of all of the animal waste, which is rich in fixed nitrogen, coming from feedlots, broiler houses, and pig farms? And what is the environmental impact of its disposal? This problem is compounded by inappropriate agricultural practice in many countries, where the plentiful supply of cheap commercial nitrogen fertilizer, plus farm subsidies, has encouraged high (and increasing) application rates. In these circumstances, only about half (at best) of the applied nitrogen reaches the crop plant for which it was intended; the rest leaches and “runs off” into streams, rivers, lakes, and finally into coastal waters. The resulting eutrophication can be detrimental to marine life. If it encroaches on drinking-water supplies, a human health hazard is possible. Furthermore, oxidation of urea and ammonium fertilizers to nitrate progressively acidifies the soil – a major problem in many agricultural areas of the world. A related problem is the emission of nitrogen oxides (NOx) from the soil by the action of microorganisms on the applied fertilizer and, if fertilizer is surface broadcast, a large proportion may be volatilized and lost as ammonia. For urea in rice paddies, an extreme example, as much as 50% is volatilized and lost to the atmosphere. And what goes up must come down; in the case of fertilizer nitrogen, it returns to Earth in the rain, often acidic in nature. This

xi uncontrolled deposition has unpredictable environmental effects, especially in pristine environments like forests, and may also affect biodiversity. Some of these problems may be overcome by more efficient use of the applied fertilizer nitrogen. A tried and tested approach (that should be used more often) is to ensure that a balanced supply of nutrients (and not simply applying more and more) is applied at the right time (maybe in several separate applications) and in the correct place (under the soil surface and not broadcast). An entirely different approach that could slow the loss of fertilizer nitrogen is through the use of nitrification inhibitors, which would slow the rate of conversion of the applied ammonia into nitrate, and so decrease its loss through leaching. A third approach to ameliorating the problems outlined above is through the expanded use of biological nitrogen fixation. It’s not likely that we shall soon have plants, which are capable of fixing N2 without associated microbes, available for agricultural use. But the discovery of N2-fixing endophytes within the tissues of our major crops, like rice, maize, and sugarcane, and their obvious benefit to the crop, shows that real progress is being made. Moreover, with new techniques and experimental approaches, such as those provided by the advent of genomics, we have reasons to renew our belief that both bacteria and plants may be engineered to improve biological nitrogen fixation, possibly through developing new symbiotic systems involving the major cereal and tuber crops. In the meantime, the major impact might be through agricultural sustainability involving the wider use of legumes, reintroduction of crop-rotation cycles, and incorporation of crop residues into the soil. But even these practices will have to be performed judiciously because, if legumes are used only as cover crops and are not used for grazing, their growth could impact the amount of cultivatable land available for food crops. Even so, the dietary preferences of developed countries (who eats beans when steak is available?) and current agricultural practices make it unlikely that the fixed-nitrogen input by rhizobia in agricultural soils will change much in the near-term future. A significant positive input could accrue, however, from matching rhizobial strains more judiciously with their host legumes and from introducing “new” legume species, particularly into currently marginal land. In the longer term, it may be possible to engineer crops in general, but cereals in particular, to use the applied fertilizer more efficiently. That would be a giant step the right direction. We shall have to wait and see what the ingenuity of mankind can do when “the chips are down” as they will be sometime in the future as food security becomes a priority for many nations. At the moment, there is no doubt that commercially synthesized fertilizer nitrogen will continue to provide the key component for the protein required by the next generation or two. So, even as we continue the discussion about the benefits, drawbacks, and likely outcomes of each of these approaches, including our hopes and fears for the future, the time has arrived to close this effort to delineate what we know about nitrogen fixation and what we have achieved with that knowledge. It now remains for me to thank personally all the authors for their interest and commitment to this project. Their efforts, massaged gently by the editorial team, have produced an indispensable reference work. The content is my responsibility and I apologize

xii upfront for any omissions and oversights. Even so, I remain confident that these volumes will serve well the many scientists researching nitrogen fixation and related fields, students considering the nitrogen-fixation challenge, and administrators wanting to either become acquainted with or remain current in this field. I also acknowledge the many scientists who were not direct contributors to this series of books, but whose contributions to the field are documented in their pages. It would be remiss of me not to acknowledge also the patience and assistance of the several members of the Kluwer staff who have assisted me along the way. Since my initial dealings with Ad Plaizier, I have had the pleasure of working with Arno Flier, Jacco Flipsen, Frans van Dunne, and Claire van Heukelom; all of whom provided encouragement and good advice – and there were times when I needed both! It took more years than I care to remember from the first planning discussions with Ad Plaizier to the completion of the first volumes in this series. Although the editorial team shared some fun times and a sense of achievement as volumes were completed, we also had our darker moments. Two members of our editorial team died during this period. Both Werner Klipp (1953-2002) and John Gallon (19442003) had been working on Volume II of the series, Genetics and Regulation of Nitrogen-Fixing Bacteria, and that volume is dedicated to their memory. Other major contributors to the field were also lost in this time period: Barbara Burgess, whose influence reached beyond the nitrogenase arena into the field of iron-sulfur cluster biochemistry; Johanna Döbereiner, who was the discoverer and acknowledged leader in nitrogen-fixing associations with grasses; Lu Jiaxi, whose “string bag” model of the FeMo-cofactor prosthetic group of Mo-nitrogenase might well describe its mode of action; Nikolai L’vov, who was involved with the early studies of molybdenum-containing cofactors; Dick Miller, whose work produced new insights into MgATP binding to nitrogenase; Richard Pau, who influenced our understanding of alternative nitrogenases and how molybdenum is taken up and transported; and Dieter Sellmann, who was a synthetic inorganic chemistry with a deep interest in how N2 is activated on metal sites. I hope these volumes will in some way help both preserve their scientific contributions and reflect their enthusiasm for science. I remember them all fondly. Only the reactions and interest of you, the reader, will determine if we have been successful in capturing the essence and excitement of the many sterling achievements and exciting discoveries in the research and application efforts of our predecessors and current colleagues over the past 150 years or so. I sincerely hope you enjoy reading these volumes as much as I’ve enjoyed producing them. William E. Newton Blacksburg, February 2004

PREFACE

Nitrogen Fixation in Agriculture, Forestry, Ecology and the Environment Most grant applications that involve basic research on biological nitrogen fixation (BNF) emphasize the economic importance of this basic biological process. But just what is the economic value of BNF? In several chapters of this volume, the authors report their estimations of the amount of nitrogen fixed biologically. For example, the amount of nitrogen fixed each year by the legume-rhizobia symbiosis is around 70 million tonnes. If we assume that at least 70% of this amount is utilised in agricultural and agroforestry systems and so replaces commercial Nfertiliser, we can estimate that around 50 million tonnes of fixed nitrogen are made available to support agricultural production. In Europe, the price of fertiliser-N to the farmer at the beginning of 2004 was 650€ per tonne, which is equivalent to US$ 800. Putting these two figures together indicates just how enormous the credit made by legume-based BNF to agriculture is. Of course, this price varies significantly in other regions of the world and is closely linked to the prices of oil and gas. This fertiliser-oil linkage is the reason that funding for both the basic and applied aspects of BNF research has increased in periods with high oil and gas prices and decreased when energy prices are moderate. Because of their importance in worldwide agriculture, the volume starts with surveys of the current uses and benefits, plus the future potential, of legumes in agricultures around the World. In Chapter 1, production and BNF of many tropical legumes, as well as fodder and green-manure legumes, such as Mucuna, are described to illustrate their potential. The "Phaseomics" consortium and its research targets are also summarized here. This consortium involves forty-six research groups working with Phaseolus sp. and is offered as a model for future collaborative research projects focussed on a single crop. Chapter 1 also suggests that, in addition to the already established legume crops, such as Phaseolus, Vigna, Arachis, Cicer, Cajanus and Mucuna species, many other tropical legumes may become regionally important crops, depending on when more research funding becomes available to develop breeding programmes that include both crop protection and crop nutrition by BNF. Soybean is one of the four most important crops worldwide and it is the number-one oil and protein supplier for animal and human nutrition. The 2002 world production of 180 million tons exceeded the combined production of all other grain legumes, such as groundnuts, beans, pigeon peas and chickpeas. Production and BNF in soybeans in the four major growing regions for this crop are described in Chapters 2-5, with Chapter 2 considering North America, Chapter 3 covering South America, Chapter 4 describing the situation in India, and Chapter 5 giving an overview of the history and current soybean cultivation in China, the original home of the soybean. The regional aspects are especially emphasized in this extensive coverage to illustrate the importance of both plant breeding (and consequent plant production) and their concurrent adaptation to regional priorities, where world

xiii

xiv market and trade regulations are becoming increasingly important. The areas planted with soybeans are 57,000 km² in India, 83,000 km² in China, 290,000 km² in South America, and 300,000 km² in North America. All other areas add only about 70,000 km2, making a global total of about 800,000 km². This area planted to soybean is more than the combined land area of France and the UK. Other crop legumes are planted on about 700,000 km², which together with the soybean area, adds up to 1.5 million km² of the Earth’s land area planted to cultivated legumes. With such a large land area dedicated to their cultivation, it is not surprising that considerable research emphasis and, in many countries strict regulatory control, is given to the preparation, production, and application of rhizobial inoculants for legumes. This is one of the few areas where research on symbiotic nitrogen fixation has directly achieved a level of economic relevance (Chapter 11). The rhizobial strain-selection programmes for soybeans and common beans in Brazil are successful examples of continuous efforts to promote inoculation with the appropriate strains and the correct techniques. The pasture and grassland areas on the five continents total about 30 million km2, but we do not have an accurate estimate of what percentage is covered with legume species. If we assume 10% coverage, they would occupy an area of 3 million km². When we add the area covered with nodulated legume trees and shrubs (Chapter 7), which is larger than previously assumed, especially in Africa, and the more than 160 species of nitrogen-fixing trees and shrubs associated with actinorhiza (Chapter 8), we can make the cautious estimate that about 10 million km² of the land area of this planet are covered with symbiotic nitrogen-fixing species. This is more than the area of the United States of America, again highlighting the importance of these symbiotic species to humankind. Among the important influences on symbiotic nitrogen fixation with these crop and related species are soil-stress factors. The major stress factors, soil water, soil pH, soil temperature, and nutrient limitations, have been studied in detail and are described in Chapter 6. The various combinations of these factors, together with the large number of host plant-microsymbiont permutations, make it impossible still to predict precisely the amount of nitrogen that will be fixed by a single plant species in any region of the World. For example, more than forty such factors, which affect nitrogen fixation by actinorhizal plants in the field, are listed in figure 3 of Chapter 8. Another fascinating research area involves the interaction of nitrogenfixing symbioses with arbuscular mycorrhizae (AM) to give a tripartite association (Chapter 10). The AM is probably the oldest symbiosis of higher plants with microbes. In some phases of the tripartite interaction, the legume-rhizobia symbiosis and the AM symbiosis not only share the same plant host, but also share signal-transduction pathways (see also M. Parniske et al., (2002) Nature). A second BNF-research area of agricultural relevance and potential involves the endophytic associations of diazotrophs with cereals and other grasses. These associations appear less formalised than the rhizobia-legume symbiosis and, unfortunately, we still do have not enough field data to quantify their economic impact. Attempts to develop such data are often frustrated by the very complicated situation that exists. For example, many of these close endophytic associations of nitrogen-fixing microbes with grasses include microbial species that cannot be

xv cultivated directly with the result that their presence can only be detected by indirect methods, such as in situ nifH mRNA expression (Chapter 9 and see also volume 5 of this series, Associative and Endophytic Nitrogen-fixing Bacteria and Cyanobacterial Associations). The volume closes with consideration of the other major processes in the Nitrogen Cycle and their relationship to nitrogen fixation. The economic impact of free-living nitrogen-fixing species is probably negligible because, before the nitrogen fixed by the bacterial cells has a chance to reach the plant, a large portion of it is likely lost and returned to the atmosphere as N2 by the combined processes of nitrification and denitrification. However, the ecological importance of the complete Nitrogen Cycle for the the quality of soils and soil functions can hardly be overestimated. Therefore, the final chapters of this volume cover these two important processes; nitrification is covered in Chapter 12 and denitrification in Chapter 13. Both of these integral parts of the Nitrogen Cycle have microorganisms as their essential components. The progress made in understanding the physiology, biochemistry, and molecular genetics that undergird these two processes is as equally impressive as that made in the field of nitrogen fixation as described in the seven volumes of this series. We could not close this volume without thanking everybody involved with its production. We are especially grateful to all the authors of this volume for contributing their experience, considerable effort, and time (over and above their regular work load) to produce interesting, up-to-date, and extensively referenced chapters. We also wish to give special thanks to Mrs. Lucette Claudet in Marburg for her dedicated work on this volume. D. Werner wants to thank the EU for the International Development Project ICA 4-CT-2001-10057, related to several chapters of this volume. Dietrich Werner Marburg, January 2005 William E. Newton Blackburg, January 2005

LIST OF CONTRIBUTORS

Rubens José CAMPO EMBRAPA-CNPSO Soja Cx. Postal 231, 86001-970, Londrina PR, Brazil. Email: [email protected]

Rosario AZCÓN Departemento Microbiologia del Suelo y Systemas Simbioticos, Estacion Experimental del Zaidin, CSIC, C/ Profesor Albareda 1, Apdo 419, E-18008 Granada, Spain. Email: [email protected]

María Jesús DELGADO IGEÑO Estación Experimental del Zaidín, CSIC, C/ Profesor Albareda 1, E-18008 Granada, Spain. Email: [email protected]

Concepción AZCÓN-AGUILAR Departemento Microbiologia del Suelo y Systemas Simbioticos, Estacion Experimental del Zaidin, CSIC, C/ Profesor Albareda 1, Apdo 419, E-18008 Granada, Spain. Email: [email protected]

Claudia FIENCKE Institut für Bodenkunde, Universität Hamburg, Allende-Platz 2, D-20146 Hamburg, Germany. Email: [email protected]

José-Miguel BAREA Departemento Microbiologia del Suelo y Systemas Simbioticos, Estacion Experimental del Zaidin, CSIC, C/ Profesor Albareda 1, Apdo 419, E-18008 Granada, Spain. Email: [email protected]

Julio C. FRANCHINI EMBRAPA-CNPSO Soja, Cx. Postal 231, 86001-970, Londrina PR, Brazil. Email: [email protected] Peter H. GRAHAM University of Minnesota, Dept. of Soil, Water, and Climate, 1991 Upper Buford Circle, St Paul, MN 55108, USA. Email: [email protected]

Eberhard BOCK Fachbereich Biologie der Universität Hamburg, Institut für Allgemeine Botanik Mikrobiologie, Ohnhorststr. 18, D-22609 Hamburg, Germany. Email: [email protected]

Mariangela HUNGRIA EMBRAPA–CNPSO Soja Cx. Postal 231, 86001-970, Londrina PR, Brazil. Email: [email protected]

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xviii Thomas HUREK Allgemeine Mikrobiologie Fachbereich 2 Biologie/Chemie, Universität Bremen, Postfach 33 04 40, D-28334 Bremen, Germany Email: [email protected] M. F. LOUREIRO UFMT/FAMEV, Av. Fernando Correa s/n Campus Universitário, 78000-900 Cuiabá, MT, Brazil. Email: [email protected] S. K. MAHNA Department of Botany, Maharshi Dayanand Saraswati University, K-22, Gandhi-Nagar, Naka Madar, Ajmer - 305001, India. Email: [email protected] IIeda C. MENDES Embrapa Cerrados. Cx. Postal 08223, 73301-970 Planaltina, DF, Brazil. Email: [email protected] William E. NEWTON Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA. Email: [email protected] Steven G. PUEPPKE Associate Dean for Research, College of Agricultural, Consumer and Environmental Sciences, 211 Mumford Hall, University of Illinois, Urbana, IL 61801, USA Email : [email protected]

Barbara REINHOLD-HUREK Allgemeine Mikrobiologie Fachbereich 2 Biologie/Chemie, Universität Bremen, Postfach 33 04 40, D-28334 Bremen, Germany. Email: [email protected] David J. RICHARDSON Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia Norwich NR4 7TJ, U.K. Email: [email protected] D.N. RODRIGUEZ-NAVARRO Centro de Investigación y Formación Agraria “Las Torres y Tomejil”, Apartado Oficial, E-41200-Alcalá del Río, Sevilla, Spain Email: dulcenombre.rodriguez@untade andalucia.es Ricardo RUSSO Institute for Forestry and Natural Resources, EARTH University, P.O. Box 4442-1000, Costa Rica Email: [email protected] José Enrique RUIZ SAINZ Departamento de Microbiologia Facultad de Biologia, Universidade de Sevilla Apartado 1095, 41080 Sevilla Spain. Email: [email protected]

xix Michael J. SADOWSKY Dept. of Microbiology and Soil Science, University of Minnesota, Borlaug Hall, 1991 Upper Buford Circle, St. Paul, MN 55113, USA. Email: [email protected] Rob J.M. van SPANNING Department of Molecular Cell Physiology, Faculty of EarTh and Life Sciences, Free University, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands. Email: [email protected] Eva SPIECK Institut für Allgemeine Botanik, Abteilung Mik robiologie, Universität Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany. Email: [email protected] Janet I. SPRENT 32 Birkhill Avenue, Wormit, Fife DD6 8PW, U.K. Email: [email protected]

Jane E. THOMAS-OATES Department of Chemistry, University of York, Heslington, York, YO10 5DD, U.K. Email: [email protected] J. M. VINARDELL Departamento de Microbiologia Facultad de Biologia, Universidade de Sevilla Apartado 1095, E-41080, Sevilla Spain. Email: [email protected] Dietrich WERNER FG Zellbiologie und Angewandte Botanik, Fachbereich Biologie, Philipps-Universität Marburg, Karl-von-Frisch-Strasse, D-35032 Marburg, Germany. E-mail: [email protected] J. C. ZHOU Department of Microbiology Huazhong Agricultural University, Shi Zi Shan Street, P.O. Box 430070, Wuhan People’s Republic of China. Email: [email protected]

Chapter 1

PRODUCTION AND BIOLOGICAL NITROGEN FIXATION OF TROPICAL LEGUMES D. WERNER Fachbereich Biologie, Fachgebiet Zellbiologie und Angewandte Botanik, PhilippsUniversität Marburg, D-35032 Marburg, Germany

1. INTRODUCTION More than 60% of grain-legume production is soybeans (Table 1). Therefore, this crop is covered by several chapters in this volume as a model for several other grain legumes in which our knowledge is much less developed due to the limited areas of production and, therefore, lower economic impact. However, for the people in many areas, especially in South America and Africa, other grain legumes are vital Table 1. World Production (FAO Yearbook, 2001)

Soybeans Groundnuts Dry beans (Phaseolus and Vigna) Dry peas Chickpeas Dry faba beans Lentils Green beans Green peas

Million tons 176.6 35.1 16.8 10.5 6.1 3.7 3.1 4.7 7.1

both for the diets of the population and for the production by small farmers. In terms of sustainable agriculture, both fodder legumes and legumes used as green 1 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 1-13. © 2005 Springer. Printed in the Netherlands.

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WERNER

manure are equally important. As examples of recent progress in understanding production and biological nitrogen fixation (BNF) in these legumes, the following species will be discussed: beans (Phaseolus sp. and Vigna), chickpea (Cicer arietinum), pigeon pea (Cajanus cajan), peanuts (Arachis sp.), Mucuna and other tropical legumes. The protein, lipid, and carbohydrate contents of grain legumes are summarized in Table 2. The protein content of most species is in the range of 20-30% in dry seeds. Only Psophocarpus tetragonolobus with 33%, Glycine max with ca. 34%, and Lupinus mutabilis with 48% are significantly beyond this range. The lipid content is very low with only 1-2% in Vigna, Phaseolus, Pisum, Cajanus, Lens, and Dolichos species. Three genera with a protein content greater than 25% have high lipid concentrations; these are 16% in Psophocarpus tetragonolobus, 18% in Glycine max, and up to 48% in Arachis hypogaea (peanuts). Table 2. Protein, lipid and carbohydrate content (%) in dry seeds of grain legumes. Modified from Souci et al., 1994.

Crop-species

Arachis hypogaea Cajanus cajan Canavalia ensiformis Cicer arietinum Dolichos lablab Glycine max Lens culinaris Phaseolus acutifolius Phaseolus lunatus Phaseolus vulgaris Pisum sativum Psophocarpus tetragonolobus Vicia faba Vigna aconitifolia Vigna angularis Vigna mungo Vigna radiata Vigna subterranea Vigna umbellata Vigna unguiculata

Protein

Lipid

Carbohydrate

25.3 20.2 25.5 19.8 22.0 33.7 23.5 24.5 20.6 21.3 22.9 33.1 23.0 23.6 20.7 23.1 24.0 19.0 21.5 23.5

48.1 1.4 2.5 3.4 1.5 18.1 1.4 1.5 1.4 1.6 1.4 16.2 2.0 1.1 1.4 1.2 1.1 7.0 0.3 1.4

8.3 47.0 50.0 41.2 50.0 6.3 52.0 65.5 45.0 40.1 41.2 30.8 55.0 56.5 56.4 41.5 43.6 54.0 60.9 41.7

2. PHASEOLUS SP. AND VIGNA SP. (BEANS) More than 200 species of the genus Phaseolus have been described (Smartt, 1988). The most important species economically are Phaseolus vulgaris var. nanus, Phaseolus coccineus, Phaseolus lunatus, Phaseolus acutifolius and Phaseolus

PRODUCTION AND BNF OF TROPICAL LEGUMES

3

semierectus. Common names for Phaseolus vulgaris are dwar f beans, bush beans and dry beans (in spanish “frielos secos”). The gene center for Phaseolus species is in Central and South America and for Vigna in Asia. The original wild form of Phaseolus vulgaris is Phaseolus vulgaris ssp. Aborigineus, which is found in moist valleys in middle America. The genus Phaseolus has a chromosome number of 2N = 22. The main gene banks are at the CIAT in Cali (Columbia), in Gatersleben (Germany), and in the Wavilov-Institute in St Petersburg (Russia). A large number of genetic variations are known that impact yield, seed colour, and early ripening. Specific morphological and physiological characters are a shallow root system, self pollination, and lightindependent development. Species can develop normally under shading conditions, making this genus suitable for agroforestry systems. The DNA content of Phaseolus, Vigna, and Psophocarpus species are summarized in Table 3. It ranges from 590 units in Phaseolus vulgaris up to 1110 in Vigna aconitifolia. Desired characteristics of new bean cultivars have been formulated by Broughton et al. (2003) and the targets and genetical components are summarized in Table 4. They include α-amylase-inhibitors, specific sugar synthesis, and Rhizobium nodulation characteristics. Table 3. DNA content of Phaseolus, Vigna and Psophocarpus species. Modified from Bennett and Leitch (2001) and Smartt (1990).

Vernacular name Common beans Lima beans Scarlet runner beans Tepary beans Long beans/cowpeas Mung beans (green gram) Urd (black gram) Adzuki beans Rice beans Moth beans Winged beans

Latin name (sub-family - Papilionoideae) Phaseolus vulgaris L. Phaseolus lunatus L. Phaseolus coccineus L. Phaseolus acutifolius A. Gray Vigna unguiculatu (L.) Walp. Vigna radiata (L.) Wilczek

Vigna mungo (L.) Hepper Vigna angularis (Willd.) Ohwi and Ohashi Vigna umbellata (Jacq.) Ohwi and Ohashi Vigna aconitifolia (Jacq.) Maréchal Psophocarpus tetragonolobus (L.) DC

IC DNA content 590 690 670 740 590 520

540 540 570 1110 780

Several other targets and plant components have been proposed by the Phaseomics Consortium, which has 43 groups worldwide (Table 5). These targets include classical physiological traits, such as nutrients and soil factors, and also new molecular genetical techniques to facilitate and improve breeding programmes. At the end, as in classical breeding, the goal is stable or adapted and productive new cultivars. It is interesting to compare these new goals with those of an FAO/IAEA coordinated research programme that ran from 1986 to 1991 (Bliss and Hardarson,

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WERNER Table 4. Targets and genes for bean breeding. Modified from Broughton et al. (2003).

Problem

Anti-nutritional factors

Flatulence Hard-to-cook Low %Ndfa Low protein seed levels Plant type Pod shatter Poor nodulation Sensitivity to Colletotrichum Seeds low in S-amino acids Susceptibility to seed-boring insects

Target

α-Amylase inhibitors, arcelins, lectins, phenolics, tannins, phytates, trypsin inhibitors Raffinose, stachyose, verbascose Cotyledonary middle lamella

Phaseolin and APA gene families Determinancy genes Pod string Legume–Rhizobium Interactions Several major loci and QTLs for resistance Phaseolin Arcelin-phytohemagglutinin-α, amylase inhibitor (APA)

Genetic component Often single genes

Genotypic variation Genotypic variation Single, complex loci fin locus st locus Many COK-4 (protein kinase), B4 cluster, and others Single, complex locus Genotypic variation Single, complex locus

1993). The main topics then were methodology to study nitrogen fixation in the field, the use of genotypic variation in different bean breeding lines and cultivars, and the classical eco-physiology of nitrogen fixation. With nine different bean lines, the amount of N2 fixed varied between 18 and 36 kg.ha–1 (kilograms per hectare) after 56 days (Kipe-Nolt and Giller, 1993). In another experimental series, the amount of N2 fixed within 60 days varied between 18 and 50 kg.ha–1 (Kipe-Nolt et al., 1993). Beans are nodulated by several species and genera of rhizobia, as summarized in Table 6. Bean species originating in Latin America harbour mainly Rhizobium etli strains in their nodules, whereas in Europe and in Africa, other Rhizobium species are present (Giller, 2001). The protein, lipid, and carbohydrate contents of seven Vigna species are summarized in Table 2. They have an important role in agriculture in many Asian and African countries. The lipid content is rather low (1-2%) with the exception of Vigna subterranean, which has a lipid concentration of 7% in dry seeds. Vigna mungo (black gram), Vigna radiata (green gram), and Vigna unguiculata (cowpea) are used as either seeds or as vegetables as green beans. The results of the Phaseomics Consortium, together with the methodologies used and developed, can be transferred in the future to several Vigna species with agricultural relevance.

PRODUCTION AND BNF OF TROPICAL LEGUMES Table 5. Phaseomics Consortium and research targets

M. Aguilar (Argentina) S. Broughton, F. De Lima (Australia) Gary Cobon (Australia) C. Atkins, P. Smith (Australia) H. Irving, M. Kelly (Australia) E. Luyten, C. Snoeck, J. Michiels, J. Vanderleyden (Belgium)

N. Terryn, M. Van Montagu (Belgium) J.-P. Baudoin, A. Maquet (Belgium) G. Volckaert (Belgium) S.-M. Tsai, D.H. Moon, A. Vettore, A.G. Simpson (Brazil) M. Melotto, L.E.A. Camargo (Brazil) A. Pedrosa, M. Guerra (Brazil) H. Antoun, S. Laberge (Canada) K. Bett, B. Tar’an, A. Vandenberg, P. Balasubramanian (Canada) K.P. Pauls, T.E. Michaels (Canada) S. Beebe, M. Blair, J. Tohme (Colombia) J. Macas, V. Nasinec (Czech Republic) J. Dolezel (Czech Republic) J.-J. Drevon (France) T. Langin, V. Geffroy (France) E. Samain, H. Driguez (France)

Effects of soil stresses on nodulation Development of a standard insect screening system for beans Proteomic analyses of beans Assimilation of fixed N

Signal transduction in host plants in response to Nod factors Isolation and characterisation of differentially expressed genes following inoculation with R. etli CNPAF512; Micro-array analysis of differently expressed P. vulgaris genes Genetic transformation of Phaseolus vulgaris and P. acutifolius Inter-specific hybridisation among Phaseolus species Genome sequencing Phytic acid and aluminium tolerance

Bean EST project – BEST Cytogenetic-based physical map of P. vulgaris Novel genes induced by PGPR, mycorrhizae and cold stress Frost tolerance Generation and analysis of ESTs Development of SSR and SNP markers for P. vulgaris and Phaseolus genetic maps Development of molecular markers in P. vulgaris Bean technology at CIAT Analysis of repetitive sequences Physical and cytogenetic mapping Tolerance of symbiotic nitrogen fixation to phosphorus deficiencies Anthracnose and beans Synthesis of sulphated pentamers Sythesis of acetyl-fucosylated pentamers) Availability of Nod-factors

5

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WERNER W. Streit (Germany) R. Papa (Italy)

R. Bollini, B. Campion, L. Lioi, A. R. Piergiovanni, F. Sparvoli (Italy) F. Shah (Malaysia) G. Hernández, M. Lará (Mexico) F. Sánchez, C. Quinto (Mexico) A. Covarrubias, J. Acosta (Mexico) J.J. Peña Cabriales (Mexico) J. Simpson, L. HerreraEstrella (Mexico) J.-P. Nap (The Netherlands) E.C. Schröder (Puerto Rico) C. Gehring, G. Bradley (South Africa) A.M. De Ron, M. Santalla (Spain) B. Broughton, X. Perret (Switzerland) R. Strasser (Switzerland) P. Gepts (USA) B. Haselkorn (USA) J. Kelly (USA) P. McClean (USA) T. McDermott, D. Bergey (USA) R. Musser (USA) S. Mackenzie (USA) D. Noel (USA) E. Triplett (USA)

Vitamins and minerals Phaseolus Italian network; Population genetics, molecular biology and plant breeding Bean storage proteins

Floral and pod development; Pest and fungal resistance Functional genomics of symbiosis Nodule organogenesis; Nod-factors and signal transduction Molecular and cellular bases of the plant adaptive responses to water deficit Involvement of trehalose in drought tolerance Development of efficient transformation systems for beans Sequencing of ESTs Gene and genomic analysis and implementation of agricultural production schemes Improvement of the P. vulgaris-Rhizobium symbiosis in Puerto Rico Stress responses – roles of natriuretic peptides Germplasm, cropping systems, bean evolution, dry bean breeding, scarlet bean breeding and snap bean breeding Rhizobial determinants of effective nodulation of beans Functional behavioral patterns of living plants Evolutionary genomics of beans Acetyl CoA carboxylase in P. vulgaris Bean breeding Genomics, especially ESTs Phosphorous metabolism in Rhizobium tropici-bean symbiosis Functional genomic analyses of bean defense responses to insect attack Genomics and transcriptomics Determinants of infection and bean nodule development Genomic micro-array hybridisation

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Table 6. Rhizobium species and strains tested in Phaseolus vulgaris-plant assays. Modified from Martinez-Romero (2003)

Rhizobium etli Rhizobium tropici R. leguminosarum bv. phaseoli R. leguminosarum bv. trifolii R. mongolense R. gallicum R. giardinii Sinorhizobium fredii Sinorhizobium meliloti Sinorhizobium terangae ORS51 Sinorhizobium arboris HAMBI 1396 Sinorhizobium kostiense HAMBI 1476 Sinorhizobium sp. BR816 Sinorhizobium sp. NGR234 Mesorhizobium loti Mesorhizobium huakuii Bradyrhizobium japonicum Bradyrhizobium sp. (Phaseolus lunatus) Bradyrhizobium elkani Bradyrhizobium sp. (Lespedeza) B038 Azorhizobium caulinodans

Nodulation + + + + + + + + + + + + + + +/– +/– + + + + +/–

N2 fixation + + + + + + –/+ + or – + or – – + –/+ + + – + – + – – –

3. ARACHIS HYPOGAEA (GROUNDNUT, PEANUT) Cultivated peanuts originated from wild peanuts (Arachis spp.) with hotspots of species richness in Mato Grosso and in Campo Grande in Brazil, where several species, such as Arachis archeri, A. setinervosa, A. marginata, A. hatschabchii, A. helodes, A. magna, A. gracilis, A. cruziana, A. pietrarellii, A. williamsii and A. monticola, are now under threat of extinction (Jarvis et al., 2003). Arachis hypogaea was directly domesticated from Arachis monticola with pods splitting open underground and having smaller seeds (Sauer, 1993). Within the species Arachis hypogaea, several subspecies, such as Arachis hypogaea ssp. fastigiata, ssp. nambyquarae, ssp. oleifera, ssp. sylvestris, ssp. communis, ssp. gigantea, ssp. hirsuta, ssp. macrocarpa and ssp. Stenocarpa, have been described (USDA-ARS, 2000; Wiersema and León, 1999). From a core collection of 1700 groundnut accessions, 900 belong to the subspecies fastigiata and about 800 to the subspecies hypogaea, based on 16 morphological and 32 agronomic characteristics (Upadhyaya, 2003). The molecular diversity in Arachis hypogaea germplasm is rather low compared to wild species of Arachis sp. However, new RAPD data revealed four distinct genetic groups within 26 accessions (He and Prakash, 1997). Compared to soybeans, regeneration and transformation systems (using

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Agrobacterium and the particle gun) are less developed in peanuts (Chandra and Pental, 2003). Worldwide about 22 million ha are cultivated with groundnuts, with about 8 million in India, 8 million in Sub-Saharan Africa and 4 million in China. About 70% of the global groundnut production is in the areas of the semi-arid tropics. The international research mandate for peanuts is at the ICRISAT (International Crops Research Institute for the Semi-Arid Tropics) in Hyderabad, India (http://www.cgiar.org/areas/ground.htm). Although a major producer of soybeans, agronomic research in the United States of America also includes work with crop rotation systems, including peanuts with cotton and soybean and corn (Jordan et al., 2002). Only 50% Ndfa for groundnut was found in field studies in Vietnam (Hiep et al., 2002). In a separate field experiment, three micronutrients, boron, zinc and molybdenum, increased pod yield of groundnuts by 20-46%, indicating that the classical limitations of groundnut production are still present (Tripathy et al., 1999). Also, physical factors, such as soil temperature, can increase source-sink economy in peanuts (Awal et al., 2003). 4. CICER ARIETINUM (CHICKPEA) World production of chickpea is around 6 million tons per year (Table 1), but in 1994, production was 8 million tons per year. The main production areas are India and Pakistan. The seed production is ca. 0.8 tons per hectare. Chickpea seeds have already been found in archaeological sites from 7400 BC. Larger seeds with some evidence for domestication have been found from 6500 BC. Chickpeas, as peas and lentils, are therefore old annual legumes from the ancient neolithic period (Zohary and Hopf, 1993). Common names for chickpeas are bengal gram in India, garbanzo in Latin America, Hamaz in the Arab world, Nohud in Turkey, and Shimbra in Ethopia (Muehlbauer and Tullu, 1997). The center of their origin is probably the Caucasus and the north of Persia. Average annual yields are ca. 600 kg.ha–1 in Africa, 500-900 kg.ha–1 in India, and 1600 kg.ha–1 in North and Central America. The germplasm of chickpea is maintained at the two international centers, ICRISAT in India and ICARDA in Syria, and also in the USDA-ARS Regional Plant Introduction Station at Pullman, U.S.A. Also in the U.S., yield ranges are very large, with around 1000-1800 kg.ha–1. About 80% of the US production is exported to India, Spain, and Mexico (http://extension.oregonstate.edu). The two main fungal diseases of chickpeas are Fusarium oxysporum sp. Ciceris, which causes the plant to wilt, and Ascochyta rubiei, which causes the Ascochyta blight with brown spots on leaves, stemps, pods and also on the seeds. Pythium sp., Rhizoctonia sp., and Sclerotinia sp. are other fungal diseases of chickpeas. In different areas of India, the amount of N2 fixation, assessed by the 15N natural abundance methods, varied between 58% Ndfa up to 86% Ndfa, with an average of 78% Ndfa (Aslam et al., 2003). The nitrogen fixed was from 90 to 180 kg.ha–1. By using 15N labelling, a fixation rate of around 90 kg N per ha was found in field-grown chickpeas in Australia (Khan et al., 2003). There is a large genomic

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heterogeneity of rhizosphere strains that nodulate chickpeas. Out of 30 isolates from chickpea nodules, five genomic species were identified; these included Mezorhizobium ciceri and Rhizobium mediterraneum (Nour et al., 1995). Plant genotypes of chickpea have been successfully selected for adaptation to soils with a high salinity and high pH, with plants nodulating even at pH 9.0 to 9.2 (Rao et al., 2002). Chickpea has, like peanut, a low level of intraspecific genetic diversity. Therefore, wild relatives from 43 other species of the genus Cicer are being used to incorporate specific morphological and physiological traits, especially resistance to abiotic and biotic stresses. This programme provides a set of very interesting future tasks for both classical and molecular plant breeders (Croser et al., 2003). 5. CAJANUS CAJAN (PIGEON PEA) Pigeon peas are nodulated by slow-growing rhizobia, which cluster into at least four different groups. One of these groups is closely related to Bradyrhizobium elkanii (Ramsubhag et al., 2002). The fixation rate of Cajanus cajan is estimated at 90 kg N per ha, which is in the middle of the range of fixation data for related systems (Table 7) (Gathumbi et al., 2002). Table 7. Average N2 fixation of legumes, estimated with the 15N natural abundance and other methods. Data from Werner (1992), Gathumbi et al. (2002), Hauser and Nolte (2002), Samba et al. (2002) and Aslam et al. (2003)

Species

Arachis hypogaea Cajanus cajan Calliandra calothyrsus Cicer arietinum Crotolaria grahamiana Crotolaria achroleuca Glycine max Lens culinaris Lupinus sp. Macroptilium atropurpureum Medicago sativa Mucuna pruriens Pisum sp. Tephrosia vogelii Trifolium sp. Vicia faba

Fixation (kg N . ha-1 per season) 100 91 24 135 142 83 100 80 150 64 250 130 150 100 250 200

Cajanus cajan is probably of African origin. It grows up to 4 m high and the pods include up to 8 seeds with ca. 20% protein. It is used as green seeds together

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with the pods, mainly in India. The yields are between 500 and 2000 kg.ha–1, which is mainly due to the large size of the plant. World production, which is statistically evaluated, is around 2 million tons per year, but the local production may be much larger. On the plant side, RAPD-PCR methods allow discrimination between Cajanus cajan from 18 other legume species, including Phaseolus vulgaris, P. coccineus, P. lunatus, Glycine max, Vicia faba, Vigna radiata, V. mungo, V. aconitifolia, Pisum sativum, Cicer arietinum, Lathyrus sativus, L. latifolius, Lens culinaris, Lupinus angustifolius, L. luteus, L. albus, Medicago sativa, and Onobrychis viciifolia (Weder, 2002). 6. MUCUNA PRURIENS (VELVET BEAN) AND OTHER LEGUMES Common names for Mucuna pruriens are “Nescafé”, mauritius bean, itchy bean, krame, picapica, chiporro, frijol, and buffalobean. Scientific synonyms are Mucuna aterrima, M. atropurpurea, M. cochinchinensis, M. cyanosperma, M. deeringiana, M. esquirolii, M. prunita, M. utilis, Stizolobium aterrinum, and Negretia pruriens. The values of the genetic similarity in Mucuna species range from 0.68 to 1.00, indicating the broad genetic base of this genus and the potential for improvements by breeding techniques (Capo-chichi et al., 2003). Seed production is in the range of 650-1300 kg.ha–1 and is supported by biological nitrogen fixation from 87-171 kg.ha–1 (Hauser and Nolte, 2002). Agriculturally, the most important use of Mucuna pruriens is as a cover crop and green manure. However, due to an unusual large number of secondary plant metabolites in the seeds, extracts from either the seeds or seed powder have documented medicinal properties, which include anabolic, androgenic, anthelmintic, anti-Parkinson, antipyretic, antispasmodic, diuretic, spermatogenic, and teratogenic effects. These effects are due to the presence of several alkaloids, saponins, and sterols, plus a high concentration (7-10%) of L-dopa. Also very important is the high concentration of serotonin. The most important medical effect of Mucuna seed components is the slowing down of the progression of Parkinson’s symptoms, the reduction of blood-sugar levels, and the stimulation of growth hormones by testosterone levels (Grover et al., 2002). Probably, Mucuna seeds should be put on the doping list by the International Olympic Committee. Chemical constituents from Mucuna sp. also have nematocidal activity (Demuner et al., 2003; Morris and Walker, 2002). Phytochemical separation of the active components indicated that the isoflavonoid, prunetin, was the most active component, causing a 70% mortality of Meloidogyne incognita. The use of Mucuna pruriens as green manure and in crop rotation with maize has proved to be especially successful. A 50-100% yield increase, compared to non-fertilized control plots, has been found in West Africa with Mucuna varieties jaspaeda, utilis, and cochinchinensis (Hauser and Nolte, 2002). These data were confirmed in Mexico, using the system Zea mays-Mucuna pruriens-Zea mays, when a 50% yield increase was found. More than a 100% increase was found with the Mucuna pruriens-Zea mays system (Eilitta et al., 2003a). A comparison of the

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effect of the white- and mottled-seeded with the black-seeded Mucuna varieties indicated superiority of the white and mottled varieties (Eilitta et al., 2003b). In principal, any number of legumes that produce a large plant biomass could be used as green manure, including the 18 tropical legume species listed in Table 8. Using legumes, such as Mucuna sp., as a cover crop has another important effect; when planted directly after the maize harvest, it out-competes weeds (http://www.forages.css.orst.edu). Furthermore, the use of Mucuna species in crop rotation influences habitat conservation in forest areas by increasing the quality of the soil and, thereby, reducing the pressure on forests in Middle and South America as well as in Africa (http://www.peregrinefund.org/maya_project). Table 8. Tropical legumes as cover crops and green manure. Modified from http://www.unu.edu/unupress, http://www.iirr.org/saem/page147-152.htm, Werner (1992), and Wang et al.( 2002).

Scientific name Arachis prostate Cajanus cajan Calopogonium mucunoides Centrosema pubescens Cicer arietinum Crotalaria juncea Desmodium uncinatum Dolichos lablab Glycine wighrii Medicago sativa Mucuna utilis Phaseolus aureus Phaseolus lathyroides Psophocarpus palustris Pueraria phaseoloides Sesbania rostrata Trifolium sp. Vigna sinensis Voandzeia subterranea

Common name Huban clover Pigeon pea Calopo Centro Chickpea San hemp Spanish clover Lablab bean Glycine (perennial soybean) Alfalfa Mucuna Green gram Phasey bean Psophocarpus Kudzu Sesbania Clover Cowpea Bambara groundnut

In areas with significant sheep, goat or cattle production, the use of legumes as green manure competes with the demand of their use as fodder legumes, but the actual use by farmers and livestock keepers in Africa is still rather low (Sumberg, 2002). The use of non-native legume species can, of course, also be a problem if they become weeds, e.g., as demonstrated for Crotalaria zanzibarica in Taiwan, which has spread within 70 years to a large number of localities on this island (Wu et al., 2003).

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The author thanks the European Union for support in the INCO DEV project “Soybean BNF Mycorrhization for Production in South Asia” ICA4-CT-200110057. REFERENCES Aslam, M., Mahmood, I. A., Peoples, M. B., Schwenke, G. D., and Herridge, D. F. (2003). Contribution of chickpea nitrogen fixation to increased wheat production and soil organic fertility in rain-fed cropping. Biol. Fertil. Soils, 38, 59-64. Awal, M. A., Ikeda, T., and Itoh, R. (2003). The effect of soil temperature on source-sink economy in peanut (Arachis hypogaea). Environ. Exp. Bot., 50, 41-50. Bennett, M. D., and Leitch, I. J. (2001). Plant DNA C-values. Retrieved September 1, 2001, from http://www.rbgkew.org.uk/cvalues/homepage.html. Bliss, F. A., and Hardarson, G. (Eds.) (1993). Enhancement of biological nitrogen fixation of common bean in Latin America. Dordrecht, The Netherlands: Kluwer Academic Publishers. Broughton, W. J., Hernández G., Blair, M., Beebe, S., Gepts, P., and Vanderleyden J. (2003). Beans (Phaseolus spp.) – model food legumes. Plant Soil, 252, 55-128. Capo-chichi, Weaver, B., and Morton, C. M. (2003). The use of molecules markers to study genetic diversity in Mucuna. Trop. Subtrol. Agroecosyst., 1, 309-318. Chandra, A., and Pental, D. (2003). Regeneration and genetic transformation of grain legumes: An overview. Curr. Sci., 84, 381-387. Croser, J. S., Ahmad, F., Clarke, H. J., and Siddique, K. H. M. (2003). Utilisation of wild Cicer in chickpea improvement – progress, constraints, and prospects. Aust. J. Agric. R., 54, 429-444. Demuner, A. J., Barbosa, L. C. D., do Nascimento, J. C., Vieira, J. J., and dos Santos, M. A. (2003). Isolation and nematocidal activity evaluation of chemical constituents from Mucuna cinerea against Meloidogyne incognita and Heterodera glycines. Quimica Nova, 26, 335-339. Eilitta, M., Sollenberger, L. E., Littell, R. C., and Harrington, L. W. (2003a). On-farm experiments with maize-mucuna systems in the Los Tuxtlas region of Veracruz, Mexico. I. Mucuna biomass and maize grain yield. Exp. Agric., 39, 5-17. Eilitta, M., Sollenberger, L. E., Littell, R. C., and Harrington, L. W. (2003b). On-farm experiments with maize-mucuna systems in the Los Tuxtlas region of Veracruz, southern Mexico. II. Mucuna variety evaluation and subsequent maize grain yield. Exp. Agric., 39, 19-27. Gathumbi, S. M., Cadisch, G., and Giller, K. E. (2002). N-15 natural abundance as a tool for assessing N-2-fixation of herbaceous, shrub and tree legumes in improved fallows. Soil Biol. Biochem., 34, 1059-1071. Giller, K. E. (2001). Nitrogen fixation in tropical cropping systems. 2nd Ed. Wallingford, U.K.: CABI Publishing. Grover, J. K., Yadav, S., and Vats, V. (2002). Medicinal plants of India with anti-diabetic potential. J. Ethnopharmacol., 81, 81-100. Hauser, S., and Nolte, C. (2002). Biomass production and N fixation in five Mucuna pruriens varieties and their effect on maize yields in the forest zone of Cameroon. J. Plant Nutr. Soil Sci., 165, 101109. He, G., and Prakash, S. (1997). Identification of polymorphic DNA markers in cultivated groundnut (Arachis hypogaea L.). Euphytica, 97, 143-149. Hiep, N. H., Diep, C. N., and Herridge, D. (2002). Nitrogen fixation of soybean and groundnut in the Mekong Delta, Vietnam. In D. Herridge (Ed.), Inoculants and nitrogen fixation of legumes in Vietnam. ACIAR Proceedings. FAO (2002). Production. Yearbook 2001. Vol. 55. Rome, Italy: Food and Agriculture Organization of the United Nations. Jarvis, A., Ferguson, M. E., Williams, D. E., Guarino, L., Jones, P. G., Stalker, H. T., et al. (2003). Biogeography of wild Arachis: Assessing conservation status and setting future priorities. Crop Sci., 43, 1100-1108.

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Jordan, D. L., Bailey, J. E., Barnes, J. S., Bogle, C. R., Bullen, S. G., Brown, A. B., Edmisten, K. L., Dunphy, E. J., and Johnson, P. D. (2002). Agron. J., 94, 1289-1294. Kahn, D. F., Peoples, M. B., Schwenke, G. D., Felton, W. L., Chen, D. L., and Herridge, D. F. (2003). Effects of below-ground nitrogen on N balances of field-grown fababean, chickpea, and barley. Aust. J. Agric. Res., 54, 333-340. Kipe-Nolt, J. A., and Giller, K. E. (1993). A field evaluation using the 15N isotope dilution method of lines of Phaseolus vulgaris L. bred for increased nitrogen fixation. Plant Soil, 152, 107-114. Kipe-Nolt, J. A., Vargas, H., and Giller, K. E. (1993). Nitrogen fixation in breeding lines of Phaseolus vulgaris L. Plant Soil, 152, 103-106. Martinez-Romero, E. (2003). Diversity of Rhizobium-Phaseolus vulgaris symbiosis: Overview and perspectives. Plant Soil, 252, 11-23. Morris, J. B., and Walker, J. T. (2002). Non-traditional legumes as potential soil amendments for nematode control. J. Nematol., 34, 358-361. Muehlbauer, I. J., and Tullu, A. (1997). Cicer arietinum L. Purdue University: NewCROP FactSHEET. Retrieved from http://www.hort.purdue.edu/newcrop/cropfactsheets/Chickpea.html. Nour, S. M., Cleyet-Marel, J. C., Normand, P., and Fernandez, M. P. (1995). Genomic heterogeneity of strains nodulating chickpeas (Cicer arietinum L.) and description of Rhizobium mediterraneum sp. nov. Intl. J. Syst. Bacteriol., 45, 640-648. Ramsubhag, A., Umaharan, P., and Donawa, A. (2002). Partial 16S rRNA gene sequence diversity and numerical taxonomy of slow growing pigeonpea (Cajanus cajan L. Millsp) nodulating rhizobia. FEMS Microbiol. Lett., 216, 139-144. Rao, D. L. N., Giller, K. E., Yeo, A. R., and Flowers, T. J. (2002). The effects of salinity and sodicity upon nodulation and nitrogen fixation in chickpea (Cicer arietinum). Ann. Bot., 89, 563-570. Samba, R. T., Sylla, S. N., Neyra, M., Gueye, M., Dreyfus, B., and Ndoye, I. (2002). Biological nitrogen fixation in Crotalaria species estimated using the 15N isotope dilution method. African J. Biotechnol., 1, 17-22. Sauer, J. D. (1993) Historical geography of crop plants – a select roster. Boca Raton, FL: CRC Press. Smartt, J. (1988). Grain legumes. Evolution and genetic resources. Cambridge, U.K.: Cambridge University Press. Souci, S. W., Fachmann, W., and Kraut, H. (1994). Die Zusammensetzung der Lebensmittel. NährwertTabellen 1986/87. 5. Aufl. Stuttgart: Medpharm Scientific Publishers. Sumberg, J. (2002). The logic of fodder legumes in Africa. Food Policy, 27, 285-300. Tripathyi, S. K., Patra, A. K., and Samui, S. C. (1999). Effect of micronutriens on nodulation, growth, yield and nutrient uptake by groundnut (Arachis hypogaea L.). Indian J. Plant Physiol., 4, 207-209. Upadhyaya, H. D. (2003). Phenotypic diversity in groundnut (Arachis hypogaea L.) core collection assessed by morphological and agronomical evaluations. Gen. Resources Crop Evol., 50, 539-550. USDA, ARS (2000). National genetic resources program. Germplasm resources information network (GRIN). Beltsville, MD: National Germplasm Resources Laboratory (http://www.ars-grin.gov.) Wang, K. H., Sipes, B. S., and Schmitt, D. P. (2002). Crotalaria as a cover crop for nematode management: A review. Nematropica, 32, 35-57. Weder, J. K. P. (2002). Identification of food and feed legumes by RAPD-PCR. Food Sci. Technol., 35, 504-511. Werner, D. (1992). Symbiosis of plants and microbes. London, U.K.: Chapman and Hall. Wiersema, J. H., and León, B. (1999). World economic plants: A standard reference. Boca Raton, FL: CRC Press. Wu, S. H., Chaw, S. M., and Rejmanek, M. (2003). Naturalized Fabaceae (Leguminosae) species in Taiwan: The first approximation. Bot. Bull. Academia Sinica, 44, 59-66. Zohary, D., and Hopf, M. (1993). Domestication of plants in the old World – The origin and spread of cultivated plants in West Asia, Europe, and the Nile Valley. Oxford, U.K.: Clarendon Press.

Chapter 2

NITROGEN FIXATION BY SOYBEAN IN NORTH AMERICA S. G. PUEPPKE College of Agricultural, Consumer, and Environmental Sciences, University of Illinois, Urbana, IL 61821, USA

1. SOYBEAN: PATHWAYS TO NORTH AMERICA AND ESTABLISHMENT AS A CROP The soybean, Glycine max (L.) Merr., is one of the world’s most important and versatile crop species. Currently grown on 76 million hectares of land on five continents (FAO, 2003), soybeans are a staple part of the human diet, particularly in Asia, where they are consumed both as a vegetable and as a variety of processed food products. Large quantities of whole soybeans, soybean oil, and soybean protein products also are traded on world markets. A member of the legume subfamily Papilionoideae, G. max is known only as a cultivated species and has never been found growing in the wild (Hymowitz, 1970; 1990). Its weedy uncultivated ancestor, Glycine soja Sieb and Zucc., is widely distributed in Asia, where it grows in fields and open areas. Soybean was domesticated from G. soja by farmers in northeastern China, who recognized its value as a food crop. The precise time frame is unknown, but soybean was likely brought into cultivation more than three millennia ago, during the Shang Dynasty (ca. 1700-1100 B.C.). It had spread to adjacent parts of China and Korea by the first century A.D. and then on to other parts of Asia, where it became an increasingly important component of the human diet. An English seaman, Samuel Bowen, introduced soybean to North America in the decade just prior to the American Revolution (Hymowitz and Harlan, 1983). He had obtained a quantity of seeds from China via England, and he gave them to an acquaintance named Henry Yonge. Yonge was the Surveyor General of the Colony of Georgia, and beginning in 1765, he grew soybeans on his plantation near Savannah. Bowen had some of the harvested seeds processed into soy sauce and 15 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 15-23. © 2005 Springer. Printed in the Netherlands.

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noodles and began experimenting with other manufacturing methods, but the undertaking ended with his death in 1777. Soybeans were reintroduced to North America on a number of occasions after Bowen, and we know that American botanists and horticulturists planted and studied them during the first half of the nineteenth century (Hymowitz, 1990). It was the year 1851, though, that marked the rebirth of investigations into the potential of soybean as a North American crop plant. This time, the seeds came from Japan via San Francisco, where Dr. Benjamin Franklin Edwards had been pressed into service to examine a group of Japanese fishermen for contagious diseases (Hymowitz, 1990). The physician found the fishermen to be healthy and, in gratitude, they presented him with a package of soybean seeds. Dr. Edwards took this unusual gift with him when he returned home to Alton, Illinois. Fortuitously, this small city lies within a vast region of farmland that is well suited to soybean production. Edwards presented the seeds to a horticulturist friend, who immediately planted them and began to distribute succeeding crops of seeds through a network of horticulturists. Within just a few years, a loosely organized group of farmers and gardeners had evaluated soybean across the U. S. Midwest as a potential forage crop for animals. As was common then, they communicated their experiences with the new crop by means of notices and articles in the popular agricultural press. The ability of legumes to fix nitrogen symbiotically had not yet been established and, if these early American soybean growers noticed root nodules, they could not have been aware of their agronomic significance. Symbiotic nitrogen fixation was initially documented by the experiments of Hellriegel and Willfarth in 1888 (Fred et al., 1932) and the soybean root-nodule organism, Bradyrhizobium japonicum, was first isolated 7 years later (Kirchner, 1895). These scientific discoveries in Europe coincided with yet another introduction of soybean into the U.S., one that would firmly establish the species as a major agricultural crop. This time, agricultural scientists deliberately brought the crop into the country, investigated its agronomic potential, and collaborated with other researchers to assure the success of the endeavor (Hymowitz, 1990). The first experiments were conducted by scientists from the New Jersey Agricultural Experiment Station at Rutgers College, who had brought seeds from Europe in 1878, multiplied them in the field, and soon distributed germplasm to agricultural experiment stations in other states. Seeds were also imported directly from Asia and, by the final decades of the nineteenth century, soybeans had become the subject of serious ongoing investigations to determine their suitability as an American crop. The artificial inoculant industry dates back to 1895, when Nobbe and Hiltner were first granted patents on the use of pure cultures of rhizobia for legume seed treatments (Fred et al., 1932). Even prior to this time, scientists had begun to determine the feasibility of ensuring nodulation and nitrogen fixation by transfer of small amounts of soil from fields in which soybeans had been grown to new fields. Cottrell and colleagues (1900) in Kansas and Lane (1900) in New Jersey were among the first to conduct such experiments with soybean in the United States and, as early as 1904, farmers had begun to offer “infected soil” for sale (Windish, 1981). Inoculation came into common practice during the early part of the twentieth century (Figure 1) and, as soybean began to establish itself commercially,

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individual states started to produce inoculants for farmers. In 1928 alone, Missouri and Wisconsin distributed 38,522 and 14,793 cultures, respectively, to soybean farmers (Fred et al., 1932).

Figure 1. An early soybean inoculant produced by the Hazelmere Bacteria Company in Bowling Green, Indiana. Adapted from Fred et al., 1932.

2. SOYBEAN PRODUCTION IN NORTH AMERICA Official records of soybean production in the United States did not appear until 1924 (Barnhart, 1954). Previously and for some time thereafter, the crop was grown on a relatively small scale, mostly as either a forage legume or a green manure to enhance soil fertility. Production initially was centered in the eastern part of the country, such that the leading states in 1919 were North Carolina, Kentucky, Mississippi, Virginia, and Alabama. Soybean production areas moved west rapidly over the ensuing five years, such that, by 1924, the leading states were Illinois, Indiana, Tennessee, North Carolina, and Missouri (Windish, 1981). Soybean first became a significant American crop during World War II, when traditional sources of vegetable oils were disrupted by the conflict. Production of soybean for oil exceeded that for hay in 1941 (Barnhart, 1954) and, by 1942, the United States overtook Manchuria as the world’s leading producer of the crop (Windish, 1981). The latter change is illustrated in Figure 2, which documents the growth and geographical redistribution of soybean production in the United States

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from 1927 to 2000. Soybean production was in its infancy in 1927 and scattered throughout the Midwest and southeast. By 1961, when soybean had become a major crop and was planted on 10.9 million hectares (FAO, 2003), production was concentrated in three Midwestern states; Illinois, Indiana, and Iowa. Smaller but significant acreages were also scattered in the south central and southeastern regions and in Minnesota, a state originally assumed to be too cold to sustain growth of crop (Cottrell et al., 1900). Huge increases in soybean production are evident in the data from 2000, which show the strengthening and continued dominance of Illinois, Indiana, and Iowa. U. S. soybeans covered an area of 29.3 million hectares in 2000, when production exceeded 75 million metric tons for the first time (FAO, 2003).

1927

MN SD NE

IA IL IN OH MO

1961

2001

Figure 2. Changes in the magnitude and geographical distribution of United States soybean production from 1927 to 2001. One dot is equivalent to 9,000 metric tons. The upper left hand map identifies the principal soybeangrowing states of the Midwest: IA = Iowa, IL = Illinois, IN = Indiana, MN = Minnesota, MO = Missouri, NE = Nebraska, OH = Ohio, SD = South Dakota. Data Source: National Agricultural Statistics Service, United States Department of Agriculture.

Mexico and Canada produce relatively minor amounts of soybeans in comparison to the United States (Figure 3). Plantings in Mexico have fluctuated significantly over the past 40 years, and production has declined over the past decade. Soybean oil is the most important plant oil produced in Mexico, but domestic soybean production does not meet domestic needs. As recently as 19961997, imports from the United States accounted for nearly half of the soybeans crushed for oil in Mexico (Juarez, 1998).

NITROGEN FIXATION BY SOYBEAN IN NORTH AMERICA

19

1200 1000

Hectares planted (X 1,000)

800

Mexico

600 400 200 0 1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 1200 1000 800

Canada

600 400 200 0 1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000

Year Figure 3. Soybean production in Mexico and Canada from 1961 to 2001. Data Source: FAOSTAT Agricultural Database of the United Nations Food and Agricultural Organization.

Soybean was grown in Canada as early as 1939, when a total of 3,960 hectares was planted (Anonymous, 1939) and test fields were sown in Western Canada as early as 1941 (Rennie and Dubetz, 1984). But it was only during the past 25 years, as early maturing varieties were developed, that Canada became a significant soybean producer. The area planted to soybeans in Canada increased from 85,800 hectares in 1961 to 279,200 hectares in 1981 to 1.1 million hectares in 2001 (FAO, 2003). About 83% of Canada’s current production areas lie in Ontario, primarily in the southern counties of the province along the north shore of the Great Lakes (Ontario Ministry of Agriculture and Food, 2003). Together, Mexican and Canadian soybean acreages are equivalent to less than 5% of the U. S. total.

20

PUEPPKE 3. MAJOR SOYBEAN CROPPING SYSTEMS

Soybean is normally grown in sequence with other crops throughout the major production areas of the American Midwest (Hoeft et al., 2000). Crop rotations of this sort are common agricultural practices and they were in place prior to widespread introduction of soybean in the middle of the twentieth century. The appearance of soybean at this time coincided with and was facilitated by another fundamental change, the replacement of animal traction with machinery. Before widespread mechanization, the rotation cycle usually encompassed four years and included oats, two years of a legume, such as either alfalfa or clover, a year of corn, and then oats again. This sequence ensured adequate feed for farm animals as well as an abundant supply of fixed nitrogen, which was not yet available as commercial fertilizer. Prior to World War II, it was possible to simply replace one of the forage legumes with soybeans for use as hay (Barnhart, 1954). With the advent of vast soybean acreages and the diminished need for animal feed, the rotation was simplified to today’s typical corn-soybean sequence. About 80% of the total cropland in the Midwest is currently managed in this way (Hoeft et al., 2000). The widespread acceptance of this practice is evident from the near identity of total corn and total soybean acreages in any given area and growing season. In 2001, for example, Illinois farmers planted 4.4 million hectares of corn and 4.3 million hectares of corn (Illinois Agricultural Statistics Service, 2002). In some of the more southern areas of the Midwest, corn often is replaced with either wheat or sorghum, but the two-year rotation with soybean is maintained (Hoeft et al., 2000). Thus, regardless of the identity of the second crop and as a rule of thumb, farmers in the American Midwest generally plant soybean every other year. 4. BIOLOGICAL NITROGEN FIXATION BY SOYBEAN IN NORTH AMERICA Biological nitrogen fixation has been assessed by a variety of protocols, each with its own inherent advantages and shortcomings (Bremner, 1977; Burris, 1974; Chalk, 1985; Danso, 1986; Danso et al., 1993; Hardarson and Danso, 1993; Knowles, 1981). 15N-based techniques have become the method of choice for legumes growing in the field (Unkovich and Pate, 2000), in part because they allow the calculation of nitrogen fixation as kg N/ha (Hardarson and Danso, 1993). Two different 15N-based protocols have been devised (Bergerson, 1980; Hardarson, 1990; Peoples et al., 1989). The natural abundance method takes advantage of the fact that the heavy isotope is relatively more prevalent in the soil than in the atmosphere. Tissues from plants with direct access to N2 from the atmosphere, thus, have lower 15N enrichment than do plants that derive nitrogen exclusively from the soil, and the resulting differences in isotope abundances can be quantified. Isotope dilution-based techniques rely on enrichment of the soil supporting the growth of nitrogen-fixing legumes and nonfixing reference plants often non-nodulating mutants - with 15N-labeled fertilizers. Because the fixing

NITROGEN FIXATION BY SOYBEAN IN NORTH AMERICA

21

plants capture gaseous N2 from the air, the ratio of 15N to 14N in their tissues decreases relative to that in the non-fixing plants and this difference allows the percentage of nitrogen derived from the atmosphere to be calculated. The literature on global nitrogen fixation is filled with uncertainty (Burns and Hardy, 1975; Hardy and Havelka, 1975; LaRue and Patterson, 1981; Smil, 2001; Sprent, 1986). This situation is partly due to differences of opinion about the most appropriate application of techniques to measure nitrogen fixation - and the interpretation of experimental results. But it also reflects a whole series of uncontrolled variables that are known to influence nitrogen fixation in the field. These include environmental factors, such as moisture, temperature, soil type, and nutrient status (especially that of mineral nitrogen), as well as plant genotype, adequacy and efficiency of nodulation (Brockwell and Bottomley, 1995), and a whole variety of agronomic practices. LaRue and Patterson (1981) summarized a series of early experiments to determine nitrogen fixation by soybean and concluded that “the upper limit for average fixation by soybeans in American agriculture is 75 kg N/ha.” This estimate is based on the assumption that soybeans growing in U.S. Midwestern fields accumulate a total of 150 kg N/ha and that 50% of the total is supplied by nitrogen fixation. This estimate agrees well with data from nine sets of field experiments, which were conducted in the 1970s and early 1980s. Three were based on isotopedilution methods and were conducted on typical silty loam soils in the Midwest. The amounts of fixed nitrogen recorded in these experiments were reasonably uniform, given the observed variations in growth conditions, and ranged from 43146 kg N/ha in eastern Nebraska (Deibert et al., 1979) to 76-152 kg N/ha in southcentral Minnesota (Ham and Cardwell, 1978) to 103 kg N/ha in east-central Illinois (Johnson et al., 1975). Unkovich and Pate (2000) revisited the issue of nitrogen fixation by legumes in the field and have summarized both a vast amount of experimental data as well as recent refinements in our understanding of measurement techniques. They conclude that average nitrogen fixation by dry land soybean supplies 100 kg N/ha on a worldwide basis and, if root biomass is included in the calculations, the amount increases to 142 kg N/ha. These figures are greater and likely more accurate than earlier estimations, but they remain subject to the considerable variability and uncertainty inherent in making generalizations about a crop cultivated under diverse agronomic regimes and across wide geographical areas. The practice of irrigation illustrates just one of these uncertainties. When soybean is grown under irrigation, levels of nitrogen fixation are elevated to 175 kg N/ha - with values approaching 250 kg N/ha if roots are included (Peoples et al., 1995). Just one factor, assured optimal moisture can, therefore, nearly double nitrogen fixation in comparison to average field conditions. 5. PERSPECTIVES If we accept the most recent figure of 142 kg N/ha, then we can estimate that, for 2001, soybeans growing in North America captured about 4.3 million metric tons of

22

PUEPPKE

nitrogen from the atmosphere and cycled it into plant organic matter. This is a significant number but, as has been pointed out (Graham and Vance, 2000; Smil, 1997), the world’s agriculture is relying less and less on biologically fixed nitrogen and more and more on synthetic fertilizer. Given the immense energy required to synthesize anhydrous ammonia (Smil, 2001), this substitution is likely not sustainable over time. Dry mass accumulation - plant growth - is often viewed as a principle driver for biological nitrogen fixation, but it would be erroneous to conclude that, as soybean yields have increased in recent decades, nitrogen fixation has just simply increased in a corresponding fashion (Unkovich and Pate, 2000). Fertilizer use is rising in cropping systems, such as the corn-soybean rotation of the U. S. Midwest, and it is well known that nodulation and nitrogen fixation are directly inhibited by combined nitrogen (Streeter, 1988). New soybean varieties are being released and we continue to know very little about rhizobia in cropped soils. These complexities both confound our understanding of biological nitrogen fixation in soybean and offer fertile ground for future investigations. ACKNOWLEDGEMENTS I thank Geetanjali Tandon and the National Soybean Research Laboratory for assistance with Figure 2. REFERENCES Anonymous (1939). Apportionment of areas, agricultural production and numbers of livestock in various countries. International yearbook of agricultural statistics 1941-42 to 1945-46 (Vol. III.). Rome, Italy: Villa Borghese. Barnhart, F. (1954). Soybeans. Cape Girardeau, Missouri: Missouri Printing and Stationery Co. Bergerson, F. J. (1980). Methods for evaluating biological nitrogen fixation. Chichester, U.K.: Wiley and Sons. Bremner, J. M. (1977). Use of nitrogen-tracer techniques for research on nitrogen fixation. In A. Ayanaba and P. J. Dart (Eds.), Biological nitrogen fixing farming systems of the tropics (pp. 335352). Chichester, U.K.: Wiley and Sons. Brockwell, J., and Bottomley, P. J. (1995). Recent advances in inoculant technology and perspectives for the future. Soil Biol. Biochem., 27, 683-697. Burns, R. C., and Hardy, R. W. F. (1975). Nitrogen fixation in bacteria and higher plants. New York, NY: Springer. Burris, R. H. (1974). Methodology. In A. Quispel (Ed.), The biology of nitrogen fixation (pp. 9-33). Amsterdam, The Netherlands: North-Holland Publishing Co. Chalk, P. M. (1985). Estimation of N2 fixation by isotope dilution: An appraisal of techniques involving 15 N enrichment and their application. Soil Biol. Biochem., 17, 389-410. Cottrell, H. M., Otis, D. H., and Haney, J. G. (1900). Soil inoculation for soy beans. Kansas State Agricultural College Experiment Station, Bulletin, 96, 97-116. Danso, S. K. A. (1986). Estimation of N2-fixation by isotope dilution: An appraisal of techniques involving 15N enrichment and their application. Soil Biol. Biochem., 18, 243-244. Danso, S. K. A., Hardarson, G., and Zapata, F. (1993). Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation. Plant Soil, 152, 25-52. Deibert, E. J., Bijeriego, M., and Olson R. A. (1979). Utilization of 15N fertilizer by nodulating and nonnodulating soybean isolines. Agron. J., 71, 717-723. FAO (2003). United Nations Food and Agricultural Organization, FAOSTAT Agricultural Database. See: http://apps.fao.org/cgi-bin/nph-db.pl?subset=agriculture. Downloaded January 10, 2003.

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Fred, E. B., Baldwin, I. L., and McCoy, E. (1932). Root nodule bacteria and leguminous plants. Madison, WI: University of Wisconsin Press. Graham, P. H., and Vance, C. P. (2000). Nitrogen fixation in perspective: An overview of research and extension needs. Field Crops Res., 65, 93-106. Ham, G. E., and Caldwell, A. C. (1978). Fertilizer placement effects on soybean yield, N2 fixation, and 32 P uptake. Agron. J., 70, 779-783. Hardarson, G. (1990). Use of nuclear techniques in studies of soil-plant relationships. Training Course Series No. 2. Vienna, Austria: International Atomic Energy Agency. Hardarson, G., and Danso, S. K. (1993). Methods for measuring biological nitrogen fixation in grain legumes. Plant Soil, 152, 19-23. Hardy, R. W. F., and Havelka, U. D. (1975). Nitrogen fixation research: A key to world food? Science, 188, 633-643. Hoeft, R. G., Nafziger, E. D., Johnson, R. R., and Aldrich, S. R. (2000). Modern corn and soybean. Champaign, IL: MCSP Publications. Hymowitz, T. (1970). On the domestication of the soybean. Econ. Bot., 24, 408-421. Hymowitz, T. (1990). Soybeans: The success story. In J. Janick and J. Simon (Eds.), Advances in new crops (pp. 159-163). Portland, OR: Timber Press. Hymowitz, T., and Harlan, J. R. (1983). Introduction of soybean to North America by Samuel Bowen in 1765. Econ. Bot., 37, 371-379. Illinois Agricultural Statistics Service (2002). See: www.agstats.state.il.us/annual/2002. Johnson, J. W., Welch, L. F., and Kurtz, L. T. (1975). Environmental implications of N fixation by soybeans. J. Environ. Qual., 4, 303-306. Juarez, B. (1998). Soy leads U. S. oilseed exports to Mexico. United States Department of Agriculture, Foreign Agricultural Service. See http://www.fas.usda.gov/info/agexporter/1998/soyleads.htm. Kirchner, O. (1895). Die wurzelknöllchen der sojabohne. Beitr. Biol. Pflanz., 7, 213-223. Knowles, R. (1981). The measurement of nitrogen fixation. In A. H. Gibson and W. E. Newton (Eds.), Current perspectives in nitrogen fixation (pp. 327-333). Amsterdam, The Netherlands: Elsevier Press. Lane, C. B. (1900). An experiment in inoculating soy bean. Twentieth Annual Report. New Jersey Agricultural Experiment Station. Camden, NJ: Sinickson Chew and Co. LaRue, T. A., and Patterson, T. G. (1981). How much nitrogen do legumes fix? Adv. Agron., 34, 15-38. Ontario Ministry of Agriculture and Food (2003). Soybeans – area and production, Ontario by county, 2001. See: www.gov.on.ca/omafra/english/stats/crops/ctysoybeans01.htm. Peoples, M. B., Faizah, A. W., Rerkasem, B., and Herridge, D. F. (1989). Methods for evaluating nitrogen fixation by nodulated legumes in the field. Canberra, Australia: Australian Centre for International Agricultural Research. Peoples, M. B., Gault, R.R., Lean, B., Sykes, J. D., and Brockwell, J. (1995). Nitrogen fixation by soybean in commercial irrigated crops of central and southern New South Wales. Soil Biol. Biochem., 27, 553-561. Rennie, R. J., and Dubetz, S. (1984). Multistrain vs. single strain Rhizobium japonicum inoculants for early maturing (00 and 000) soybean cultivars: N2 fixation quantified by 15N isotope dilution. Agron. J., 76, 498-502. Smil, V. (1997). Some unorthodox perspectives on agricultural biodiversity - the case for legume culture. Agric. Ecosyst. Environ., 62, 135-144. Smil, V. (2001). Enriching the earth. Cambridge, MA: MIT Press. Sprent, J. I. (1986). Benefits of Rhizobium to agriculture. Trends Biol., 4, 124-129. Streeter, J. (1988). Inhibition of legume nodule formation and N2 fixation by nitrate. Crit. Rev. Plant Sci., 7, 1-22. Unkovich, M. J., and Pate, J. S. (2000). An appraisal of recent field measurements of symbiotic N2 fixation by annual legumes. Field Crops Res., 65, 211-228. Windish, L. G. (1981). The soybean pioneers. Henry, IL: M and H Printing.

Chapter 3

THE IMPORTANCE OF NITROGEN FIXATION TO SOYBEAN CROPPING IN SOUTH AMERICA M. HUNGRIA1, J. C. FRANCHINI1, R. J. CAMPO1 AND P. H. GRAHAM2 1 2

Embrapa Soja, Cx. Postal 231, 86001-970, Londrina, PR, Brazil. University of Minnesota, Department of Soil, Water, and Climate, 1991 Upper Buford Circle, St Paul, MN 55108, USA.

1. INTRODUCTION Although soybean (Glycine max L. Merr.) is a relatively recent crop introduction to South America, the region currently accounts for about 45% of world soybean production. This paper provides a brief overview of both soybean production in this region and some of the problems that have been overcome in reaching current production and yield levels. 2. TAXONOMY, ORIGINS, AND IMPORTANCE OF SOYBEAN Hymowitz and Newell (1980) divide the genus Glycine into the subgenera, Glycine and Soja. The former genus includes seven wild perennial species, most of which are indigenous to Australia (Brown et al., 1985), whereas the latter includes the cultivated soybean, Glycine max (L.) Merr., and its annual wild counterpart, G. soja Sieb. and Zucc. G. soja, with twice the nucleotide diversity of G. max (Cregan et al., 2002) occurs throughout northern, north-eastern and central China and in adjacent areas of the former USSR, Korea, Japan, and Taiwan. Hymowitz and Singh (1987) infer more than one domestication event in the Shang dynasty (ca. 1700-1100 BC) with the resulting emergence of soybean as a domesticated crop in central and northern China during the period from 1100-700BC. Movement of the crop into India, Nepal, Burma, Thailand, Indochina, Korea, Japan, Malaysia, Indonesia, and the Philippines occurred by the first millenium AD (Smartt and 25 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 25-42 . ©2005 Springer. Printed in the Netherlands.

26

HUNGRIA, FRANCHINI, CAMPO AND GRAHAM

Hymowitz, 1985) and was accompanied by change in several host genetic traits that affected symbiosis with rhizobia (Pulver et al., 1982; Devine, 1984). Europe first learned of soybean in 1712 through the writings of the German scientist, Englebert Kaempfer, with seeds sent by missionaries stationed in China and subsequently planted in the Jardin des Plantes, Paris, in 1740 (Probst and Judd, 1973). Glycine max was introduced into the USA in 1804, probably as a result of seed interchange between the USA and France (Hymowitz and Newell, 1980), and initially attracted more interest for its potential as a forage species (Probst and Judd, 1973). Significant production of soybean in the United States and Europe dates only from the beginning of the twentieth century (Piper and Morse, 1923; Gray, 1936; Morse, 1950; Hymowitz, 1970), with major germplasm collection and evaluation only initiated between 1927 and 1931 (Probst and Judd, 1973). Even today, the Glycine germplasm maintained in the USDA Germplasm Resource Information Network (http://www.ars.grin.gov) includes only 18,765 accessions of G. max, 1,117 of G. soja, and approximately 1,000 accessions of related Glycine spp. Also of concern is the very narrow genetic base used until recently in the majority of soybean-breeding programs. Thus, Delannay et al. (1983) noted that 10 introductions provided 80% of the northern US gene pool, whereas only 7 introductions contributed the same percentage to the southern US gene pool. Soybean today is one of the most important and extensively grown crops in the world. It accounts for 29.7% of the world’s processed vegetable oil and is a rich source of dietary protein both for the human diet and for the chicken and pork industries (Graham and Vance, 2003). Isoflavones from soybean may reduce the risks of cancer and lower serum cholesterol (Molteni et al., 1995; Kennedy, 1995). Soybean is also used as a milk substitute in weaning foods and baking and in ink and biodiesel fuels (Anonymous, 2000; 2001; Graham and Vance, 2003). Although soybean production continues to grow worldwide (Figure 1), with an estimated production in 2002/2003 of 183.28 Tg, a major part of this production comes from only four countries: the USA, Brazil, Argentina, and China (Figure 2). Mercosur, the common market agreement signed by Brazil, Argentina, Paraguay, and Uruguay in 1991, is today responsible for about 45% of world soybean production (Uruguay accounts for 0.03% of the production). Production has increased significantly over the last few years (Figure 3), even though that from the USA has declined from 47% of world production in 1999/2000 to 39% in 2001/2002 (CONAB, 2002a). Bolivia, a partner of Mercosur in the Andean region, now plants 680,000 ha annually with an estimated production of 1.3 Tg in 2002/2003 (USDA, 2002a). Export of soybean from South America continues to grow and to capture most of the increases in world soybean demand with the most dramatic increase being observed in Argentina (USDA, 2002b). Soybean was probably introduced into Brazil in 1882, in the State of Bahia, as Soja hispida (Glycine soja) and Soja ochroleuca (D´utra, 1882; 1899). A few studies were subsequently undertaken at the experimental-station level, but it was not until the 1940s that soybean was grown commercially in the southern region of Brazil. A significant expansion of soybean production in this region occurred in the 1960s but, even as late as 1975, Brazil produced only 11.24 Tg of soybean annually. This. situation started to change in the 1970s, with the expansion of crop production

NITROGEN FIXATION AND SOYBEANS IN SOUTH AMERICA

27

Million tons 200

180

160

140 2000

2001

2002

2003

Figure 1. World soybean production in the last three years, with projection for 2002/2003 (CONAB, 2002a).

O thers China India USA Argentina

EC Paraguay Brazil Figure 2. Contribution of different countries to world soybean production (estimated as 183.28 Tg) in 2002/2003 (CONAB, 2002a).

28

HUNGRIA, FRANCHINI, CAMPO AND GRAHAM

Figure 3. Annual soybean production (Tg) in the United States, and in the four main producers in South America: Brazil, Argentina, Paraguay and Bolivia (CONAB, 2002a; 2002c; 2003; USDA, 2002a).

into the "Cerrados”, an edaphic savanna occupying 207 million ha and 25% of the land area of Brazil. The Cerrados are distinct in their soil chemical properties and environment (Goedert, 1985), necessitating the use of varieties that had longer juvenile periods, were aluminum tolerant, and were calcium-use efficient (Spehar, 1995). Initial yields were low but, as appropriate technologies were developed for the area, steadily improved. In 2002/2003, the three main states of this ecosystem, which are Mato Grosso, Mato Grosso do Sul, and Goiás (including the Federal District) cultivated 7.80 million ha and produced 23,329 Tg of soybean, with a yield average (2,991 kg ha-1) greater than the national mean (2,765 kg ha-1). A 10.6% increase in production area is expected in 2002/2003 (CONAB, 2003). Soybean was known in Argentina in 1880, but the first field experiments there date from around 1908, and only 1,315 ha were planted in 1941/42. As in Brazil, significant expansion in the area cropped to soybean in Argentina dates to the mid 1970s with 169,400 ha cropped in 1972/73, 9.5 million ha in 1988/89, and 12.3 million ha in 2002 (Saumell, 1977; Miró, 1989; Anonymous, 2001). Soybean was introduced into Paraguay in the 1920s with seeds from the United States, Argentina, and Japan, but commercial expansion did not occur until the 1970s (Alvarez, 1989). Importation of Brazilian cultivars adapted to the tropics facilitated crop improvement and continues today (Oliveri et al., 1981). Breeding for improved productivity and for adaptation to the shorter day length, high temperature, and edaphic constraints common in the Cerrados have been a major part of soybean improvement in Brazil and have resulted in numerous varietal releases. Traditional breeding programs, which include both hybridization of selected parents in single, three/way or multiple combinations and selection by pedigree, mass selection or single/seed descent, have been used with both increased

NITROGEN FIXATION AND SOYBEANS IN SOUTH AMERICA

29

crop yield and yield stability emphasized. Selection for a long juvenile period (Toledo et al., 1994; Spehar, 1995) now allows soybean to be produced even in the state of Roraima, localized between 1ºS and 2ºN latitudes. Another decision, made by the National Soybean Commission in the 1960s, was that biological nitrogen fixation (BNF) was an important trait that needed to be considered in breeding activities. Most Brazilian cultivars of this period were derived from North American genotypes, and differences between them in relation to their symbiotic performance had already been identified (Döbereiner and Arruda, 1967; Vargas et al., 1982). Selection of parental lines active in symbiotic N2 fixation with Bradyrhizobium was emphasized in the early breeding activities, but attention to this trait has declined in recent years. A consequence has been a decline in the symbiotic capacity of recently released cultivars (Bohrer and Hungria, 1998; Hungria and Bohrer, 2000). Nicolás et al. (2002) studied the genetics of nodulation and nitrogen fixation in Brazilian cultivars with contrasting symbiotic efficiency, and reported narrow-sense heritability (hn2) estimates of from 39% to 77% for nodulation and plant growth under low fixed-N soil conditions. These values are high compared to others reported in the literature. Simple sequence repeat (SSR) markers related to symbiotic performance in soybean have also been identified (Nicolás, 2001) and can now be used in the search for cultivars with higher biological N2 fixation capacity. 3. BIOLOGICAL NITROGEN FIXATION The area cropped with soybean in Brazil increased from 702,000 ha in 1940/41 to 17.95 million ha in 2002/2003. Soybean now accounts for 42% of national agricultural production and 16% of gross internal product (CONAB, 2002a; 2002b; 2003). The increase in area has been paralleled by a more than four-fold increase in national mean yield over the period from 1968 to the present (Figure 4). Argentina, Paraguay, and Bolivia report similar changes. As a consequence of the higher yields, plant fixed-N demands have also more than doubled in the last 40 years. A program to select more efficient and competitive strains has been in operation in Brazil since the 1960s and continues to ensure that soybeans in Brazil can satisfy their fixed-N requirements through N2 fixation (Vargas and Hungria, 1997; Hungria et al., this volume). Bacterial strains are selected for use in commercial inoculant production only after extensive field testing in the main soybean-production areas. Four strains are currently recommended for use; Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 (=29w), since 1979, and B. japonicum SEMIA 5079 (=CPAC 15) and SEMIA 5080 (=CPAC 7), 1992 (Vargas and Hungria, 1997; Hungria and Vargas, 2000; Hungria et al., this volume). Each of these strains can fulfill the crops need for fixed-N at yields greater than 4,000 kg ha-1. They are provided free to inoculant producers, and all inoculants must carry two of the four strains. Their genetic relatedness with other well-studied Bradyrhizobium strains is shown on Figure 5.

30

HUNGRIA, FRANCHINI, CAMPO AND GRAHAM

3,000

Yield

2,500

Area

20,000 15,000

1,500

10,000

Area

Yield

2,000

1,000 5,000 500 0

0 1968

1978

1988

1998

2003

Figure 4. Change in mean yield (kg ha-1) and area (x 1,000 ha) cropped with soybean in Brazil over the last 45 years (Anonymous, 2000; CONAB, 2002c; 2003).

G e n e tic D is ta n c e 97

98

99

100

B . elka nii

S E M IA 5 0 1 9

B . elka nii

U S D A 31

B . elka nii

U S D A 76 T

B . elka nii

U S D A 94

B . elka nii

S E M IA 5 8 7

B . ja p o nicu m

S E M IA 5 0 8 0

B . ja p o nicu m

U S D A 12 2

B . ja p o nicu m

S E M IA 5 8 6

B . ja p o nicu m

U S D A 11 0

B . ja p o nicu m

U S D A 13 6

B . ja p o nicu m

S E M IA 5 0 8 5

B . ja p o nicu m

S E M IA 5 6 6

B . ja p o nicu m

USD A 6T

B . ja p o nicu m

U S D A 12 3

B . ja p o nicu m

S E M IA 5 0 7 9

Figure 5. Genetic relatedness among soybean Bradyrhizobium strains recommended for use in commercial inoculants in Brazil (SEMIA 587, SEMIA 5019, SEMIA 5079, SEMIA 5080) and Argentina (SEMIA 5085). After Chueire et al. (2003).

NITROGEN FIXATION AND SOYBEANS IN SOUTH AMERICA

31

Although more than 90% of the area cropped with soybean in Brazil today has been inoculated before, and generally contains naturalized rhizobia, yield increases averaging 4.5% have been obtained when soils containing 103 or more cells g-1 soil were reinoculated (Hungria et al., this volume). Table 1 shows some of these results. This finding contrasts with those of studies in the USA (Thies et al., 1991) and the difference in response warrants further detailed studies in both regions. Currently, re-inoculation is practiced by about 60% of farmers in Brazil (ANPI, Associação Nacional dos Produtores de Inoculante, personal communication) and by 50% of those in Argentina (Dr. Enrique R. Moretti, Laboratórios BIAGRO, Argentina, personal communication). As a result, the number of inoculant doses sold each year continues to increase in both countries (Figure 6). This situation is, again, in marked contrast to the USA, where the use of inoculants is a common practice in just 15% of the soybean-production area but, in Canada, about 60% of the farmers have adopted inoculation (Dr. R. Stewart Smith, Nitragin, USA, personal communication). In Argentina, the strain recommended by INTA (Instituto Nacional de Tecnologia Agropecuaria) is B. japonicum E109 (=SEMIA 5085, =USDA 138), and its relatedness with other strains is shown in Figure 5. Table 1. Effects of reinoculation and of addition of N-fertilizer on soybean yield (kg ha-1) in soils with established populations of soybean bradyrhizobia (> 103 cells g-1 soil). After Hungria et al. (2001).

Treatment

Non-inoculated Non-inoculated + N-fertilizer2 Inoculated with efficient strains CV (%)

South Region Londrina Ponta Grossa 3,836 a1 2,697 b 3,434 b 2,872 ab

4,025 a 9.9

Cerrados Goiânia Planaltina 2,341 a 2,483 a 2,432 a 2,660 a

2,912 a

2,462 a

8.1

8.8

3,119 a 7.0

Values followed by the same letter, in the column, do not show statistical difference (Duncan, p≤0.05). 2 200 kg of N ha-1 as urea, split at sowing and at early flowering.

1

In contrast to their Mercosur neighbours, Paraguay has had no strain-selection program and just 15-20% of the soybean crop is inoculated. There is also no quality-control program in the country and locally produced and foreign inoculants used over the past two decades have often been of poor quality. Inoculants now sold in Paraguay come from Argentina, Brazil, and Uruguay. Mercosur legislation permits inoculants produced in one country to be sold in another, provided they carry strains recommended by local rhizobiologists. Despite the less-than-ideal situation, nodulation occurs without inoculation in most areas of Alto Paraná and Itapúa, where 80% of soybean production occurs (Figueredo, 1998). Symbiotic effectiveness varies from site to site but, in a recent survey, some isolates collected in the field were outstanding symbiotic performers, a first step for the identification of strains for use in commercial local inoculants (Chen et al., 2002).

32

HUNGRIA, FRANCHINI, CAMPO AND GRAHAM

15

Argentina

Brazil

12 9 6 3 0 70/71 75/76 80/81 85/86 90/91 93/94 95/96 97/98 98/99 99/00 00/01 01/02 Figure 6. Units of soybean inoculant (x106) sold in Argentina and Brazil. (Dr. Enrique R. Moretti, Laboratórios BIAGRO, Dr. Solon Araújo, ANPI, and Dr. Laura Machado Ramos, Ministério da Agricultura, Pecuária e Abastecimento, personal communication).

4. ECONOMIC IMPORTANCE OF BIOLOGICAL NITROGEN FIXATION (BNF) IN SOUTH AMERICA Economic returns from soybeans dependent on BNF in South America are outstanding. Brazil is used here as an example. For each 1,000 kg of soybean produced, approximately 80 kg of N (65 kg N allocated to seeds, which have ca. 40% protein, and 15 kg N left in roots, stems, and leaves) are required. When the national mean yield in Brazil in 2002/2003 of 2,765 kg ha-1 is considered, the fixedN requirement would be slightly more than 220 kg ha-1. Where this is supplied through BNF, N losses are minimal. In contrast, the N-fertilizer-use efficiency of plants in the tropics is rarely as much as 60%, meaning that such plants would require ca. 360 kg N or 800 kg of urea (at 45% N) to achieve equivalent yields. At US$0.20 kg-1 of urea, the cost of applying N-fertilizer would be ca. US$160 ha-1, or in excess of US$2.87 billion nationally (for 17.95 million ha). Because Brazilian transportation costs are very high, but total production costs are 40% lower than in the United States (CONAB, 2002a; 2002b), it is evident that BNF plays a key role in lowering Brazilian production costs. Both Unkovich and Pate (2000) and Giller (2001) suggest a potential for N2 fixation by nodulated soybeans of 360-450 kg of N ha-1. Thus, there is still significant room in Brazil for increased yield without N fertilization. The situation in the other countries in South America is similar. The data available on the quantification of BNF under field conditions in South America is limited but the results from studies undertaken in Brazil are shown in Table 2. In these studies, the contribution of BNF to N accumulation among cultivars released in recent years is appreciably higher than was achieved in 33 commercial soybean fields in Australia. For these crops, with a mean % of N

NITROGEN FIXATION AND SOYBEANS IN SOUTH AMERICA

33

derived from fixation estimated at 53%, the contribution of BNF to N accumulation was 178 kg N ha-1 (Unkovich and Pate, 2000). Table 2. Contribution of biological N2 fixation to soybean production in some field experiments performed in Brazil.

Site

Condition1

Yield (kg ha-1)

Londrina

Londrina

Brasilia (Cerrados) Uberlândia (Cerrados)

2

%N from BNF

250

CT

5,410

ca. 300

84.6 (15N) 88.8 (Ndifference) 74.1

NT CT

5,890 4,380

NT

4,510

CT

4,128

ca. 300 251 (ureide) 242 (δ15N) 282 (ureide) 294 (δ15N) 292

81.0 72.5 (ureide) 69.3 (δ15N) 80.4 (ureide) 84.3 (δ15N) 87

NT CT

4,383 1,641

245 109

72 79.8

CT

3,482

234

88

NT

3,293

208

94

Rio de Janeiro2

Londrina, (Paraná)

kg of N2 fixed ha-1

Method

Reference

15

N isotope dilution, Ndifference Ureides

Boddey et al. (1984) Zotarelli (2000)

Ureides and δ15N

Zotarelli (2000)

Ureides

Hungria et al. (2003)

N isotope dilution

Boddey et al. (1990) Reis et al. (2002)

15

Ureides

1 CT, conventional tillage; NT, no-tillage system. Plants grown in the field in concrete cylinders and evaluated 92 days after planting.

There is increasing pressure for farmers that grow high-profit crops to purchase N-fertilizer. However, as discussed by Hungria et al. (this volume), starter-N doses as low as 20-40 kg of N ha-1 may decrease both nodulation and BNF under Brazilian conditions, with no benefits to yield (Crispino et al., 2001; Mendes et al., 2000; Hungria et al., 2001). Indeed, in more than 50 experiments, where inoculation and fertilization with 200 kg of N ha-1 have been compared (split application of N at sowing and flowering), no increase in yield due to N-fertilizer use has been reported. The results of one of these experiments are shown in Table 3. Similarly, there were no benefits when N-fertilizer was applied at a rate of 400 kg N ha-1, split across ten application times (Nishi and Hungria, 1996; Hungria et al., 1997, 2001; Crispino et al., 2001; Loureiro et al., 2001). It is important to emphasize that the application of only 30 kg of N ha-1 would imply a cost of US$13.3 ha-1 to the farmer and of US$239 million to the country per growing season, which would certainly decrease the competitiveness of the national product.

34

HUNGRIA, FRANCHINI, CAMPO AND GRAHAM

Table 3. Effects of inoculation and application of N fertilizer on nodule number (NN) and dry weight (NDW), root dry weight (RDW), yield and total seed N (TNG) of soybean, cultivar UFV-18. Experiment performed in Jaciara, Mato Grosso, in a soil with 105 soybean bradyrhizobia g-1. Modified from Loureiro et al. (2001).

Treatment1

NN (n° pl-1) 38 c 40 bc

Non-inoculated Non-inoculated + 200 kg N SEMIA587 54 a SEMIA5080 49 ab SEMIA587+5080 45 abc SEMIA587+5080 46 abc + 30 kg N ha-1 at sowing SEMIA587+5080 50 ab + 50 kg N ha-1 at pre-flowering SEMIA587+5080 47 abc + 50 kg N ha-1 at pod filling CV (%) 16.0 1

NDW (mg pl-1) 1,22 bc 0,97 c

RDW (g pl-1) 1,40 b 1,67 ab

Yield (kg ha-1) 2,498 b 2,668 ab

TNG (kg Nha-1) 156 b 166 ab

1,97 a 1,54 ab 1,55 ab 1,24 bc

1,49 ab 1,54 ab 1,49 ab 1,72 a

2,640 ab 2,612 ab 2,781 a 2,535 b

167 ab 169 ab 179 a 163 ab

1,22 bc

1,50 ab

2,580 ab

168 ab

1,60 ab

1,66 ab

2,527 b

169 ab

25.6

14.1

7.2

9.2

Peat inoculants containing 108 cells g-1 were applied at a rate of 500 g 50 kg-1 seeds, as a slurry with 300 ml of a 10% sucrose solution; N fertilizer applied as urea.

5. CROP MANAGEMENT IN SOUTH AMERICA Soybean is grown in South America as a main summer cash crop. In Argentina, Paraguay, Uruguay and the southern region of Brazil, a rotation with a winter crop, usually wheat (Triticum aestivum), is also adopted by many farmers. Benefits to the succeeding crop are well documented, with yields after soybean considerably higher than after the other main summer crop, maize (Zea mays). Results from one such experiment in the state of Rio Grande do Sul, Brazil, are shown in Table 4. Most of the benefits are certainly due to BNF, which results in residues with a higher level of N for the following crop. As shown in Table 5, wheat yields in plots previously used for inoculated soybean were higher than those in plots with uninoculated soybean, with or without N-fertilizer (Nishi and Hungria, 1996; Hungria et al., 1997, 2001). This increased yield and consequent profit should be considered in calculating economic benefits from the inoculation of soybean. Where rainfall is not a limiting factor in winter, other crops may be used in crop rotation with soybean, bringing benefits in terms of disease control and improvement of the soil’s physical and chemical properties, among others. Unfortunately, many of these crops are not highly profitable in the short term, with farmers often reluctant to use them. Plant species successful in crop rotation with soybean include radish (Raphanus sativus),

NITROGEN FIXATION AND SOYBEANS IN SOUTH AMERICA

35

blue lupin (Lupinus angustifolium), black oat (Avena strigosa), white oat (Avena sativa), crotalaria (Crotalaria juncea), millet (Penisetum glaucum), clovers (Trifolium spp.), common vetch (Vicia sativa), canola (Brassica napus), and linho (Linum usitatissimum) (Torres et al., 1996, 2001; Gaudencio, 1999; Calegari, 2000; Gaudencio and Costa, 2001). Crop-rotation experiments have shown the benefits to soybean yield (Table 6; ranging from 12-64%), with the lowest yields usually from continuous double crop wheat-soybean or fallow-soybean. In the Brazilian Cerrados, where irregular rainfall limits activities to one crop per year, alternative crops, including millet and forage peanut (Arachis pintoi) have been evaluated, shown to grow well, and adopted by some farmers. Table 4. Mean yield of wheat as a function of the previous crop. After Wiethölter (2000).

Year

Previous crop

Soybean

Difference

Maize kg ha-1

1993 1994

3,039 2,632

2,744 2,320

kg ha-1 295 312

1995 1996 19971 1998 Mean1

3,418 4,591 1,998 3,031 3,342

2,995 4,172 1,062 2,463 2,939

423 419 936 568 403

1

Excess rainfall in 1997 decreased yield.

Table 5. Residual effects of soybean inoculation in the summer on wheat yield (kg ha-1) in the winter. After Hungria et al. (2001). Treatment Non-inoculated Non-inoculated + 200 kg N ha-1, split at sowing and at early flowering stage Inoculated with efficient strains CV (%)

1994 1,827 ab1 1,484 b

1998 2,028 a 2,219 ab

2,000 a 12.7

2,449 b 9.3

Values followed by the same letter in a column do not show statistical difference (Duncan, p≤0.05).

1

36

HUNGRIA, FRANCHINI, CAMPO AND GRAHAM Table 6. Compilation of data of experiments with crop-rotation systems in southern Brazil.

Nº of Location systems tested 12 Campo Mourão 8 Londrina

Best systema

Ca/Mz/Rd/Sb Wt/Sb/Wt/Sb Rd/Mz/Oa/Sb Wt/Sb/Wt/Sb Londrina Oa/Sb Londrina Oa/Sb Londrina Sb/Oa/Mz-Mi Sb/Wt/Sb/Wt Campo Lp/Mz/Oa/Sb Mourão Wt/Sb/Wt/Sb Passo Fundo Oa+Cl/Sb/Oa+ Cv/Mz/Wt/Sb Guarapuava Cv/Mz/Wt/Sb

4 4 4 8 9 7 a

Worst systema

Year

Wt/Sb

Yield ReferIncrease encesb (%) 2000/01 12 1

Wt/Sb

1999/00 17

1

Fallow/Sb Fallow/Sb Sb/Wt

1985/86 24 1987/88 24 1997/98 24

2 2 3

Wt/Sb

1997/98 32

3

Oa/Sb/Wt/Sb 1992 64 Oa/Sb Li/Sb/Cv/Mz 1984/89 15 Wt/Sb

4 5

Ca, canola; Mz, maize; Rd, radish; Sb, soybean; Wt, wheat; Oa, black oat; Mi, millet; Lp, lupin; Cl, clover; Cv, common vetch; Li, linho. b 1, Gaudencio and Costa (2001); 2, Torres et al. (1996); 3, Gaudencio (1999); 4, Fontaneli et al. (2000); 5, Santos et al. (1998).

No-till (NT) soil management systems are particularly important in many areas of South America, where soils are commonly of low organic matter content, structurally fragile, and of low fertility. In NT, seeds are sown directly through the residue of the previous crop, so protecting the soil against erosion by water, maintaining soil structure, stability and moisture content, helping in the regulation of soil temperature and, with time, increasing soil organic matter content (Derpsch et al., 1991). In 1999, the world area under NT was ca. 45.5 million ha (Table 7), but is increasing substantially. By 2002, the NT area in the USA was ca. 26.3 million ha, with 13 million ha of soybean (Anonymous, 2002), whereas the NT area in Brazil was 17.356 million ha (with 4.9 million of that in the Cerrados) in 2000/2001 (FEBRAPDP, 2003) and is ca. 20 million ha in 2002/2003, which represents half of the cropped area in Brazil. For BNF in soybean, NT results in higher number of bradyrhizobia in the soil; better nodulation, with nodulation extending lower in the soil profile; and higher BNF rates and yields in relation to conventional tillage (CT). Bradyrhizobial strain diversity in the soil is also enhanced (Ferreira et al., 2000; Hungria, 2000; Hungria and Vargas, 2000). Without placing an economic value on biodiversity, Calegari (2000) estimated that a farmer adopting NT for a 50-ha area of soybean in southern Brazil would gain US$8,902 as the result of higher yield and lower costs for maintenance, fuel, labor work, and fertilizers. This result is somewhat at variance with studies in the

NITROGEN FIXATION AND SOYBEANS IN SOUTH AMERICA

37

northern USA, where cooler early-season soil temperatures and high soil-moisture content under conservation tillage can delay nodulation, result in enhanced root rot, and contribute to lowered flavonoid exudation from the root, limiting nodulation (Zhang et al., 1996; 2002; Estevez de Jensen et al., 2002). Table 7. Estimates of world area under no-till management systems in 1998/99. After Derpsch (2000).

Country United States Brazil Argentina Canada Paraguay Bolivia Uruguay Others Total

Area (1,000 ha) 19,347 11,200 7,270 4,080 790 200 50 2,596 45,533

In an 18-year field trial, Torres et al. (2001) found that CT (one disc plough and two light-disc harrow) soybean yield was higher for the first four years, but thereafter soybean yield was always higher under NT (Figure 7). Water and soil temperature conditions are always better under NT. In the first years under NT, some physical (e.g., soil density), chemical, and biological (e.g., lower nutrient 4,000

Conventional tillage

No tillage

3,000

2,000

1,000

99/00

98/99

97/98

96/97

95/96

94/95

93/94

92/93

91/92

90/91

89/90

88/89

87/88

86/87

85/86

84/85

83/84

82/83

0

Figure 7. Soybean yield (kg ha-1on left axis) under no-tillage and conventional systems in different agricultural years. Field trial performed in an oxisol of Londrina, State of Paraná. After Torres et al. (2001).

38

HUNGRIA, FRANCHINI, CAMPO AND GRAHAM

content due to microbial immobilization) changes may result in lower yields (Derpsch et al., 1991; Torres et al., 1996, 2001). Later yields are higher because of both improvements in soil physical properties, which include soil aggregation, and increases in total soil C and labile soil C in the microbial biomass, so regulating mineral cycling (Hungria, 2000; Franchini et al., 2002; Brandão-Junior et al., 2002). Higher soybean yields after the first years under NT were also found in another 11year study in southern Brazil, where soybean yield was sometimes twice that under non-conservation soil management systems (Figure 8).

Figure 8. Soybean yields after 11 years under seven different systems of soil preparation. Experiment performed in Londrina, State of Paraná. Columns with the same letter are not statistically different (Duncan, p≤ 0.05). After Torres et al. (2001).

6. FINAL CONSIDERATIONS Soybean production in South America has expanded enormously in recent years, both in area and in crop yield. In Brazil, improvements in crop yield and crop economics have been affected very markedly by a research and extension effort focused on BNF. Lines that are active in symbiosis with Bradyrhizobium have traditionally been favored in breeding. A strong and consistent effort is expect ed to select the most efficient inoculant strains and to market high quality inoculants. The result is that more than 50% of Brazilian farmers practice re-inoculation and quantification experiments in Brazil have shown that the crop derives 69-94% of its fixed-N need from symbiosis. In contrast, van Kessel and Hartley (2000) suggest that, for trials undertaken mainly in the USA and Australia, the percentage of fixedN derived from BNF has declined since 1984 and is now only 54%; however, only

NITROGEN FIXATION AND SOYBEANS IN SOUTH AMERICA

39

about 15% of farmers in the USA inoculate soybean. Some contrast between the South American and US systems of production is inevitable but any comparison should highlight problems to be avoided in South America. There is pressure to increase production in the South America by using fertilizer N. The consequences of such a change, however, need to be considered very carefully. For example, a recent commentary by Randall (2001) suggests that the more intensive corn and soybean production practiced in the mid-western USA is not sustainable. Randall points out several concerns: (i) a dependence of this production system on federal assistance; (ii) significant problems of soil erosion in the region; and (iii) declines in soil and water stewardship. Soybean growers in South America need both to maintain their focus on biological nitrogen fixation as an economic fixed-N source for this crop and to ensure that land quality is maintained (or improved) by continued study of soil quality and the factors affecting it. ACKNOWLEDGEMENT The research group in Brazil is supported by CNPq (PRONEX and 520396/96-0). The authors dedicate this chapter to the memory of Raul Martínez Lalis (Nitragin Argentina) and Bernardo Leicach (Laboratórios BIAGRO) who played an important role in improving the quality of the inoculants in Mercosur. REFERENCES Alvarez, E. R. (1989). La soja en el Paraguay, retrospectiva y perspectiva. In II Curso Internacional sobre Producción de Soja (pp. 551-563). Encarnación, Brazil: Ministerio de Agricultura y Ganaderia. Anonymous (2000). Anuário Brasileiro da Soja 2000. Santa Cruz do Sul, Brazil: Gazeta. Anonymous (2001). Anuário Brasileiro da Soja 2001. Santa Cruz do Sul, Brazil: Gazeta. Anonymous (2002). Agropecuária-Tele News at http://orbita.starmedia.com/~telenews/agricultura.html. Retrieved December 10, 2002. Boddey, R. M., Chalk, P. M., Victoria, R. L., and Matsui, E. (1984). Nitrogen fixation by nodulated soybean under tropical field conditions estimated by the 15N isotope dilution technique. Soil Biol. Biochem., 16, 583-588. Boddey, R. M., Urquiaga, S., and Neves, M. C. P. (1990). Quantification of the contribution of N2 fixation to field-grown grain legumes - a strategy for the practical application of 15N isotope dilution technique. Soil Biol. Biochem., 22, 649-655. Bohrer, T. R. J., and Hungria M. (1998). Avaliação de cultivares de soja quanto à fixação biológica do nitrogênio, Pesq Agropec Bras., 33, 937-953. Brandão Junior, O., Souza, R. A., Crispino, C. C., Franchini, J. C., Torres, E., and Hungria, M. (2002). Variação da biomassa microbiana em sistemas de manejo e rotação de culturas envolvendo a soja. In O.F. Saraiva and C.B. Hoffman-Campo (Eds.), Resumos do II Congresso Brasileiro de Soja e Mercosoja (p. 250). Londrina, Brazil: Embrapa Soja. Brown, A. H. D., Grant, J. E., Bhurdon, J. J., Grace, J. P., and Pullen, R. (1985). Collection and utilization of wild perennial Glycine. In R. Shibles (Ed.), World Soybean Research Conference III (pp. 345-352). Boulder, CO: Westview Press. Calegari, A. (2000). Plantas de cobertura e rotação de culturas no sistema plantio direto. In M. Veiga (Coord.), Memorias de la V Reunión Bienal de la Red Latinoamericana de Agricultura Conservacionista (CD ROM). Florianópolis, Brazil: EPAGRI/FAO. Chen, L. S., Figueredo, A., Villani, H., Michajluk, J., and Hungria, M. (2002). Diversity and symbiotic effectiveness of rhizobia isolated from field-grown soybean in Paraguay. Biol. Fert. Soils, 35, 448457.

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Chueire, L. M. O., Bangel, E., Mostasso, F. L., Campo, R. J., Pedrosa, F. O., and Hungria, M. (2003). Classificação taxonômica das estirpes de rizóbio recomendadas para as culturas da soja e do feijoeiro baseada no seqüenciamento do gene 16s RNA. Rev. Bras. Ci. Solo, 27, 833-840. CONAB (Companhia Nacional de Abstecimento) (2002a). Retrieved December 10, 2002, from http//www.conab.gov.br/politica_agricola/ConjunturaSemanal/Semana19a2308/Soja-19a2308.doc. CONAB (Companhia Nacional de Abstecimento) (2002b). Retrieved December 10, 2002, from http//www.conab.gov.br/politica_agricola/ConjunturaSemanal/Semana2810a0111/ConjunturaSeman aSoja13.doc. CONAB (Companhia Nacional de Abstecimento) (2002c). Retrieved December 10, 2002, from www.conab.gov.br/politica_agricola/ Safra/Quadro9.xls. CONAB (Companhia Nacional de Abastecimento) (2003). Retrieved March 20, 2003, from www.conab.gov.br. Cregan, P., Zhu, Y., Song, Q., and Nelson, R. (2002). Sequence variation, haplotype analysis and linkage disequilibrum in cultivated and wild soybean populations. In First International Conference on Legume Genomics and Genetics: Translation to Crop Improvement, June 2-6 (p. 11). MinneapolisSt Paul, MN. Crispino, C. C., Franchini, J. C., Moraes, J. Z., Sibaldelle, R. N. R., Loureiro, M. F., Santos, E. N., et al. (2001). Adubação Nitrogenada na Cultura da Soja (Comunicado Técnico, 75). Londrina, Brazil: Embrapa Soja. Delannay, X., Rodgers, D. M., and Palmer, R. G. (1983). Relative genetic contributions among ancestral lines to North American soybean cultivars. Crop Sci., 23, 944-949. Derpsch, R. (2000). Expansión mundial de la siembra directa y avances tecnológicos. In M. Veiga (Coord.), Memorias de la V Reunión Bienal de la Red Latinoamericana de Agricultura Conservacionista (CD ROM). Florianópolis, Brazil: EPAGRI/FAO. Derpsch, R., Roth, C. H., Sidiras, N., and Kopke, U. (1991). Controle da erosão no Paraná, Brasil: Sistemas de cobertura do solo, plantio direto e preparo conservacionista do solo (Sonderpublikation der GTZ, 245). Eschborn, Germany: GTZ. Devine, T. E. (1984). Genetics and breeding of nitrogen fixation. In M. Alexander (Ed.), Biological Nitrogen Fixation: Ecology, Technology and Physiology (pp. 127-154). New York, NY: Plenum Press. Döbereiner, J., and Arruda, N. B. (1967). Interrelações entre variedades e nutrição na nodulação e simbiose da soja. Pesq. Agropec. Bras., 2, 475-487. D'utra, G. (1882). Soja. J Agricultor VII, 185-188. D’utra, G. (1899). Nova cultura experimental da soja. Bol. Inst. Agron. Campinas, 10, 582-587. Estevez de Jensen, C., Percich, J. A., and Graham, P. H. (2002). Integrated management strategies of bean root rot with Bacillus subtilis and Rhizobium in Minnesota. Field Crops Res. 74, 107-115. FEBRAPDP (Federação Brasileira de Plantio Direto na Palha) (2003). Retrieved February 24, 2003, from http://www.febrapdp.org.br/pd_area_estados.htm. Ferreira, M. C., Andrade, D. S., Chueire, L. M. de O., Takemura, S. M., and Hungria, M. (2000). Effects of tillage method and crop rotation on the population sizes and diversity of bradyrhizobia nodulating soybean. Soil Biol. Biochem., 32, 627-637. Figueredo, A. (1998). Fijación Biológica de Nitrógeno. Asunción, Paraguay: Universidad Nacional de Asuncion. Dirección de Investigación, Postgrado y Relaciones Internacionales. Fontaneli, R. S., Santos, H. P., Voss, M. and Ambrosi, I. (2000). Rendimento e nodulação de soja em diferentes rotações de espécies anuais de inverno sob plantio direto. Pesq. Agropec. Bras., 35, 349355. Franchini, J. C., Crispino, C. C., Souza, R. A., Torres, E., and Hungria, M. (2002). Biomassa microbiana e emissão de CO2 em sistemas de manejo do solo e rotação de culturas. In O. F. Saraiva and C. B. Hoffman-Campo (Eds.), Resumos do II Congresso Brasileiro de Soja e Mercosoja (pp. 133). Londrina, Brazil: Embrapa Soja. Gaudencio, C. A. (1999). Sistema de rotação de espécies perenes e anuais para recuperação biológica de Lassolos Roxos eutróficos e integração agropecuária, na Região Meridional. In Resultados de Pesquisa da Embrapa Soja – 1998 (pp. 181-210; Documentos, 125). Londrina, Brazil: Embrapa Soja. Gaudencio, C. A., and Costa, J. M. (2001). Rotação de culturas anuais no Planalto Meridional Paranaense. In Resultados de Pesquisa da Embrapa Soja -2000, Manejo do Solo e Plantas Daninhas (pp. 19-25; Documentos, 161). Londrina, Brazil: Embrapa Soja. Giller, K. E. (2001). Nitrogen fixation in tropical cropping systems. Wallingford, UK: CABI Publishing.

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Goedert, W. J. (1985). Solos dos Cerrados, Tecnologias e Estratégias de Manejo. Planaltina, Brazil: Embrapa Cerrados. Graham, P. H., and Vance, C. P. (2003). Legumes: Importance and constraints to greater utilization. Plant Physiol., 131, 872-877. Gray, G. E. (1936). All About the Soya Bean. London, UK: John Bale. Hungria, M. (2000). Características biológicas em solos manejados sob plantio direto. In M. Veiga (Coord.), Memorias de la V Reunión Bienal de la Red Latinoamericana de Agricultura Conservacionista (CD ROM). Florianópolis, Brazil: EPAGRI/FAO. Hungria, M., and Bohrer, T. R. J. (2000). Variability of nodulation and dinitrogen fixation capacity among soybean cultivars. Biol. Fert. Soils, 31, 45-52. Hungria, M., and Vargas, M. A. T. (2000) Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil. Field Crops Res., 65, 151-164. Hungria, M., Campo, R. J., Franchini, J. C., Chueire, L. M. O., Mendes, I. C., Andrade, D. S., et al. (2003). Microbial quantitative and qualitative changes in soils under different crops and tillage management systems in Brazil. In Annals of Technical Workshop on Biological Management of Soil Ecosystems for Sustainable Agriculture (CD ROM). Londrina, Brazil: Embrapa Soja/FAO. Hungria, M., Campo, R. J., and Mendes, I. C. (2001). Fixação Biológica do Nitrogênio na Cultura da Soja (Circular Técnica, 13). Londrina, Brazil: Embrapa Soja/Embrapa Cerrados. Hungria, M., Loureiro, M. F., Mendes, I. C., Campo, R. J., and Graham, P. H. (2005). Inoculant preparation, production and application. This volume. Hungria, M., Vargas, M. A. T., Campo, R. J., and Galerani, P. R. (1997). Adubação Nitrogenada na Soja? (Comunicado Técnico, 57). Londrina, Brazil: EMBRAPA-CNPSo. Hymowitz, T. (1970). On the domestication of soybean. Econ. Bot., 24, 408-421. Hymowitz, T., and Newell, C. A. (1980). Taxonomy, speciation, domestication, dissemination, germplasm resources and variation in the genus Glycine. In R.J. Summerfield and A.H. Bunting (Eds.), Advances in Legume Science (pp. 251-264). Kew, UK: Royal Botanic Gardens. Hymowitz, T., and Singh, R. J. (1987). Taxonomy and speciation. In J.R. Wilcox (Ed), Soybeans, Improvement, Production and Uses. 2nd Edit. (pp. 23-48). Madison, WI: Amer. Soc. of Agronomy. Kennedy, A. R. (1995). The evidence for soybean products as cancer preventative agents, J. Nutr., 125, S733-S743. Loureiro, M. F., Santos, E. N., Hungria, M., and Campo, R. J. (2001). Efeito da Reinoculação e da Adubação Nitrogenada no Rendimento da Soja em Mato Grosso (Comunicado Técnico, 74). Londrina, Brazil: Embrapa Soja. Mendes, I. C., Hungria, M., and Vargas, M. A. T. (2000). Resposta da Soja à Adubação Nitrogenada na Semeadura, em Sistemas de Plantio Direto e Convencional na Região do Cerrado (Boletim de Pesquisa, 12). Planaltina, Brazil: Embrapa Cerrados. Miró, D. A. (1989). La expansión del cultivo de soja y su impacto en el comercio exterior argentino. In IV Conferência Mundial de Investigación en Soja (pp. 18-27). Buenos Aires, Argentina: Associación Argentina de la Soja. Molteni, A., Brizio-Molteni, L., and Persky, V. (1995). In vitro hormonal effects of soybean isoflavones, J. Nutr., 125, S751-S756. Morse W. J. (1950). History of soybean production. In K.L. Markley (Ed), Soybeans and Soybean Products, vol. 1 (pp. 3-59). New York, NY: Interscience Publ. Inc. Nicolás, M. F. (2001). Fixação Biológica do Nitrogênio e Nodulação em Cultivares de Soja Brasileiras: Controle Genético e Mapeamento dos QTLs que Controlam esses Caracteres. Curitiba, Brazil: Universidade Federal do Paraná (Ph.D. Thesis). Nicolás, M. F., Arias, C. A. A., and Hungria, M. (2002). Genetics of nodulation and nitrogen fixation in Brazilian soybean cultivars. Biol. Fert. Soils, 36, 109-117. Nishi, C. Y. M., and Hungria, M. (1996). Efeito na reinoculação na soja [Glycine max (L.) Merrill] em um solo com população estabelecida de Bradyrhizobium com as estirpes SEMIA 566, 586, 587, 5019, 5079 e 5080, Pesq. Agrop. Bras., 31, 359-368. Oliveri, N. J., Perucca, C. E., and Morel, F. (1981). Evolución de Cultivares de Soja (Glycine max L. Merr.) de Origen Brasileño. Instituto Nacional de Tecnología Agropecuaria - Estación Experimental Agropecuaria INTA, Misiones, (Informe Técnico, 35). Piper, C. V., and Morse, W. J. (1923). The Soybean. New York, NY: McGraw-Hill.

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Probst, A. H., and Judd, R. W. (1973). Origin, history and develop, and world distribution. In B.E.Caldwell (Ed.) Soybean, Improvement, Production and Uses (pp. 1-15). Madison, WI: American Society of Agronomy. Pulver, E. L., Brockman, F., and Wein, H. C. (1982). Nodulation of soyabean cultivars with Rhizobium spp. and their response to inoculation with R. japonicum. Crop Sci., 22, 1065-1070. Randall, G. W. (2001). Intensive corn soybean production is not sustainable. Retrieved December 10, 2002, from www.extension.umn.edu/extensionnews/2001/IntensiveCornSoybeanAgriculture.html. Reis, E. H. S., Lara Cabezas, W. A. R., Alves, B. J. R., and Caballero, S. U. (2002). Suplementação de nitrogênio mineral na cultura da soja (Glycine max) estabelecida em sistema plantio direto e convencional. In O.F. Saraiva and C.B. Hoffman-Campo (Eds.), Resumos do II Congresso Brasileiro de Soja e Mercosoja (pp. 215). Londrina, Brazil: Embrapa Soja. Santos, H. P., Lhamby, J. C. B., and Wobeto, C. (1998). Efeito de culturas de inverno em plantio direto sobre a soja cultivada em rotação de culturas. Pesq. Agropec. Bras., 33, 289-295. Saumell, H. (1977). Soja, Información Técnica para su Mejor Conocimiento y Cultivo. Buenos Aires, Argentina: Editorial Hemisferio Sur. Smartt, J., and Hymowitz, T. (1985). Domestication and evolution of grain legumes. In R.J. Summerfield and E.H. Roberts (Eds.), Grain Legume Crops (pp. 37-72). London, UK: Collins Professional and Technical Books. Spehar, C. R. (1995). Impact of strategic genes in soybean on agricultural development in the Brazilian tropical savannahs. Field Crops Res., 41, 141-146. Thies, J. E., Singleton, P. W., and Bohlool, B. B. (1991). Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes. Appl. Environ. Microbiol., 57, 19-28. Toledo, J. F. F., Almeida, L. A., Kiihl, R. A. S., Panizzi, M. C. C., Kaster, M., Miranda, L. C., and Menosso, O. G. (1994). Genetics and breeding. In Tropical Soybean Improvement and Production (pp. 19-36). Rome, Italy: FAO. Torres, E., Norman, N., and Garcia, A. (1996). Sucessão soja/aveia preta. In Resultados de Pesquisa de Soja – 1990/91, vol. 2, pp. 337-341 (Documentos 99). Londrina, Brazil: EMBRAPA-CNPSo. Torres, E., Saraiva, O. F., Gazziero, D. L. P., Pires, M. S. and Loni, D. A. (2001). Avaliação de sistemas de preparo do solo e manejo do plantio direto envolvendo sucessão e rotaçao de culturas. In Resultados de Pesquisa da Embrapa Soja - 2000, Manejo do Solo e Plantas Daninhas (pp. 9-14). Londrina, Brazil: Embrapa Soja. USDA (United States Department of Agriculture) (2002a). Retrieved December 10, 2002, from http://ffas.usda.gov/wap/circular/2002/02-12/Wap%2012-02.pdf. USDA (United States Department of Agriculture) (2002b). Retrieved December 10, 2002, from http://ffas.usda.gov/oilseeds/circular/2001/01-12/full.pdf. Unkovich, M. J., and Pate, J. S. (2000). An appraisal of recent field measurements of symbiotic N2 fixation by annual legumes. Field Crops Res., 65, 211-228. Van Kessel, C., and Hartley, C. (2000). Agricultural management of grain legumes; has it led to an increase in nitrogen fixation? Field Crops Res., 65, 165-181. Vargas, M. A. T., and Hungria, M. (1997). Fixação biológica do N2 na cultura da soja. In M. A. T. Vargas and M. Hungria (Eds.), Biologia dos Solos de Cerrados (pp. 297-360), Planaltina, Brazil: Embrapa-CPAC. Vargas, M. A. T., Peres, J. R. R., and Suhet, A. R. (1982). Fixação de nitrogênio atmosférico pela soja em solos de cerrado. Inf. Agrop., 8, 20-23. Wiethölter, S. (2000). Características químicas de solo manejado no sistema plantio direto. In M. Veiga (Coord.), Memorias de la V Reunión Bienal de la Red Latinoamericana de Agricultura Conservacionista (CD ROM). Florianópolis, Brazil: EPAGRI/FAO. Zhang, F., Charles, T. C., Pan, B. and Smith, D. L. (1996). Inhibition of the expression of Bradyrhizobium japonicum nod genes at low temperatures. Soil. Biol. Biochem., 28, 1579-1583. Zhang, H., Prithiviraj, B. Souliemanov, A., D'Aoust, F., Charles, T. C., Driscoll, B. T., and Smith, D. L. (2002). The effect of temperature and genistein concentration on lipo-chitooligosaccharide (LCO) production by wild type and mutant strains of Bradyrhizobium japonicum. Soil Biol. Biochem., 34, 1175-1180. Zotarelli, L. (2000). Balanço de Nitrogênio na Rotação de Culturas em Sistemas de Plantio Direto e Convencional na Região de Londrina, PR. Seropédica, Brazil: Universidade Federal Rural do Rio de Janeiro (M.Sc. Thesis).

CHAPTER 4

PRODUCTION, REGIONAL DISTRIBUTION OF CULTIVARS, AND AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA S. K. MAHNA Department of Botany, Maharshi Dayanand Saraswati University of Ajmer, India

1. INTRODUCTION AND HISTORICAL BACKGROUND Soybean (Glycine max (L.) Merrill) has been one of the man’s principle food plants for ages. Soybean has been cultivated since 2800 B.C. in China (more than 5000 years ago), which is also considered to be the center of origin for soybean. Soybean ranks first among the major oilseed crops of the world and has now found a prominent place in India. India is the fifth largest producer of soybean after the United States, Brazil, China, and Argentina (Table 1). Soybean cultivation in India started long ago but its successful cultivation has increased over last two decades. This increased cultivation has revolutionized the rural economy and improved the socio-economic status of farmers. Soybean farming made an unprecedented expansion in India between 1969 and 1996 when an annual growth rate of 15-20% was achieved. Presently, the area covered by soybean cultivation is around 5.7 x 106 hectares (ha) as recorded for the winter 2002-03 (SOPA Report, 2002-03). Soybean is grown mainly in Madhya Pradesh, Maharashtra, Rajasthan, and in small pockets in other states, like Uttar Pradesh, Andhra Pradesh, Punjab, Tamil Nadu, Uttaranchal, Gujarat, Karnataka, and Chhattisgarh. Despite being an exotic crop to India, soybean occupies a vital place in its agriculture, edible-oil economy, and foreign exchange. Tiwari et al. (1994) identified suitable soybean varieties for the non-traditional regions of India and demonstrated soybean to be a successful crop in northern, eastern, and southern regions of the country. Soybean cultivation in the Indian subcontinent dates back to 1000 A.D. The crop was introduced from China through the ‘silk route’ in the Himalayan mountain ranges running across the Tibetan plateau and through the North-East regions (Assam). Around the same time, the crop was introduced to Central India from Japan, South 43 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 43-66. © 2005 Springer. Printed in the Netherlands.

MAHNA 44

Table 1. World Production of Soybeans. Area in million hectare; Yield in tonnes/hectare; Production (Prod.) in million tonnes. After Oilseed World Market and Trade (USDA, 1999) and Agriculture Statistics at a Glance, Director of Economic and Statistical Ministry of Agriculture, GOI (as given by Paroda, 1999).

Brazil

United States

6.20

7.47

11.80

25.55

Area

0.99

1.81

1.77

2.27

2.53

Yield

5.20

11.20

13.22

26.80

64.84

Prod.

1.20

0.46

5.86

7.10

8.35

13.00

27.97

Area

1.12

2.23

3.37

1.15

2.35

1.67

2.33

2.62

Yield

152.26

7.17

2.90

1.44

6.72

16.00

13.80

30.00

74.22

Prod.

70.65

5.61

1.25

0.54

6.30

7.40

8.00

12.90

28.66

Area

(2.23)

1.51

2.64

3.26

0.90

2.53

1.73

2.40

2.62

Yield

157.70

8.40

3.30

1.74

5.70/5.90

18.70

13.00

31.00

75.03

Prod.

1998-1999

China

5.23

3.44

2.70

6.41

(2.19)

1997-1998

Argentina

0.34

2.25

6.47

69.34

1996-1997

India

1.20

1.22

131.58

Country

Paraguay

5.29

(2.08)

1.15

Others

63.19

European Union

Total/(Average)

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA

45

China and South-East Asia (Hymowitz and Kaizuma, 1981). Black-seeded soybean, under the names Bhatt, Bhatmash, or Kalitur, has since been cultivated for many years in the hilly areas of Assam, Bengal, Manipur, in the hills of Khasi and Naga, and at 6000 ft. elevation in the Kumaun regions as well as the Garhwal hills (Anonymous, 1956). Hooker (1879) clearly described soybean in his book “Flora of British India, Vol. II” and Williams (1932) mentioned the cultivation of soybean in his book “Flora India, Vol. II”. Between the years 1885-1904, attempts were made to cultivate soybean at Nagpur (Maharashtra), Madras (Tamil Nadu), Lahore (Former Punjab of undivided India), Bombay, Pune (Maharashtra), and Surat (Gujarat), but these attempts were not encouraging. However, the work carried out between 1910-1935 in Madhya Pradesh, Assam, Orissa, Bihar, Gujarat, and Uttar Pradesh paved the way for successful cultivation in India (Kaltenbach and Legros, 1936; Kale, 1936). A varietal trial, which was comprised of 33 varieties of Manchurian and Chinese origin, was conducted in 1933 and resulted in the establishment of soybean cultivation in Central India in 1936. Two varieties, “Otootan” (black seeded) and “Easy Cook” were promoted between 1935 and 1952 in addition to the country type (desi) Punjab white. Other varieties from the USA, viz., “Harbinsoy”, “Chiquito”, “George Washington”, “Mammoth Yellow”, and “Biloxi” were also evaluated (Tiwari et al., 1999). In India, large-scale cultivation of soybean started in 1964, using yellow-seeded high-yielding soybean exotic varieties (“Bragg”, “Clark-63”, and “Lee”) received from USA. They were tested at Jawaharlal Nehru Krishi Vishwa Vidhyalaya (JNKVV) Jabalpur, Madhya Pradesh, and almost concurrently at Govind Ballabh Pant (GBP) University of Agriculture and Technology, Pantnagar, Uttaranchal, under a major collaboration with the USA (Paroda, 1999). Subsequently, in the midseventies, cultivation practices that were suitable for Indian conditions (Saxena et al., 1971) and several new soybean varieties were introduced (Saxena and Pandey, 1971; Singh and Saxena, 1975). Since then, both the area cultivated and the production of soybean have increased until what was a marginal crop is now a major cash crop and is recognised as the miracle “golden bean” of the 20th century (Singh et al., 2001). 2. ALL-INDIA AREA COVERAGE, PRODUCTIVITY, AND PRODUCTION OF SOYBEAN BETWEEN 1970-2003 Soybean has seen phenomenal growth in both area and production in India (Paroda, 1999). In 1970-71, soybean was grown on 32,000 ha with a production of 14,000 tonnes and a productivity of 426 kg/ha. The area of soybean cultivation, production, and productivity has gradually increased over the years. In 1977-78, a six-fold increase from 32,000 to 195,000 ha in cultivation area occurred with a thirteen-fold increase from 14,000 tonnes to 183,000 tonnes in production and a two-fold increase from 426 to 940 kg/ha in productivity. In 1987-88, the area of soybean cultivation increased almost eight times, to 1,543,000 ha with a significant increase in production, to 898,000 tonnes, however, productivity decreased significantly to 582 kg/ha. This rapid growth in area of soybean cultivation continued in the next decade. In 1997-98, the area under soybean cultivation increased to 5.99 x 106 ha and the

46

MAHNA

yield increased to 1,079 kg/ha, resulting in a record production of 6.463 x 106 tonnes (Agricultural Statistics at a Glance, 2001). During the winter season of 1999-2000, the area of soybean cultivation in India was only 5.645 x 106 ha, which was about 11.15 %, lower than in 1998-99 (SOPA Report, 2000 -01). In the subsequent two seasons, an increasing trend in the area of soybean cultivation occurred. During 2001-02, the area increased to 6.0021 x 106 ha, which is about 6.32% higher than in 1999-2000 and 3.27% higher than in 2000-01. In the next winter season (2002-03), a decrease of 5.44% in cultivation area was recorded. Production and productivity data for the last five seasons (1998-2003) reveal that a maximum productivity and production of 928 kg/ha and 5.90 x 106 tonnes, respectively, were recorded in the 1998-99 season. However, in the four subsequent growing seasons, a variable reduction in both productivity and production was recorded (Table 2). 3. ALL-INDIA STATE-WISE AREA COVERAGE, PRODUCTIVITY, AND PRODUCTION OF SOYBEAN All-India state-wise area coverage of soybean during the winters of 1998 through 2003 are summarized in Table 2, which also indicates variability in area, production, and productivity in the different states of India. Area coverage, productivity, and production trends over the last five years are critically analysed below for three main soybean-producing states of the country. 3.1. Madhya Pradesh In India, Madhya Pradesh is recognised as the “Soya state” because of its significant contribution in soybean production (70-80%). In Madhya Pradesh, area coverage, yield estimate, and production of soybean have shown a relatively decreasing trend during the last four seasons (1999-2003). The maxima in yield (945 kg/ha), production (4.18 x 106 tonnes) as well as of covered area (44.3 million ha) were all observed in 1998-99, whereas the minima in yield (855 kg/ha), production (3.28 x 106 tonnes), and covered area (38.3 million ha) all occurred in 2002-03 (SOPA Reports, 2002-03). The sowing status of soybean for the year 2000-01 also revealed that, in the Mandsaur and Neemach districts, cotton and maize sowing was the alternative for soybean. Soybean cultivation at present occurs in the 45 districts of seven divisions of the state: Jabalpur, Sagar, Rewa, Indore, Ujjain, Gwalior, and Bhopal (Table 3). 3.2. Maharashtra In the state of Maharashtra, area covered under soybean cultivation and production was rather variable during last five years (1998-2003). Data for the season 2002-03 indicated that area coverage and production were 1.22 x 106 ha and 1.215 x 106 tonnes, respectively, which were about 24% and 27.7%, respectively, higher in the previous season. Not much variability was observed in the yield of soybean over the last five years.

44.3

-

10.9

6.17

0.50

0.75

0.15

0.22

0.63

63.5

Chhatisgarh (2)

Maharashtra (3)

Rajasthan (4)

Uttar Pradesh (5)

Karnataka (6)

Gujrat (7)

Andhra Pradesh (8)

Other States (9)

Total/ Average*

A

Madhya Pradesh (1)

State (S No.)

0.34 59.0

928*

0.33

0.09

0.42

0.22

5.37

10.4

-

41.8

Prod

540

1495

600

555

434

870

958

-

945

Pdty

1998-1999

56.5

0.15

0.36

0.31

0.40

0.28

4.84

11.3

-

38.8

A

895*

530

525

550

500

500

738

940

-

916

Pdty

1999-2000

50.50

0.08

0.19

0.17

0.20

0.74

3.57

10.62

-

35.53

Prod.

58.1

0.02

0.28

0.28

0.65

0.15

6.24

10.2

-

40.3

A

912*

525

600

500

535

600

800

980

-

926

Pdty

2000-2001

53.04

0.010

0.168

0.140

0.350

0.092

4.950

10.02

-

37.30

Prod.

60.0

0.75

-

-

-

-

6.54

9.82

0.54

42.4

A

900*

550

-

-

-

-

740

970

560

920

Pdty

2001-2002

54.00

0.41

-

-

-

-

4.84

9.52

0.30

38.93

Prod.

Table 2. All India State Wise Area Coverage and Yield Estimates of Soybeans during 1998-2003. Area (A) in 105 hectares; Productivity (Pdty) in kg/hectare; Production (Prod.) in 105 tonnes. After SOPA Reports (2000-2001; 2001-2002; and 2002-2003).

56.8

2.06

-

-

-

-

4.17

12.2

-

38.4

A

876*

-

-

-

-

-

712

998

-

855

Pdty

2002-2003

49.71

1.798

-

-

-

-

2.967

12.15

-

32.79

Prod.

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA 47

48

MAHNA

The sowing status in the state of Maharashtra, as provided in the SOPA Report (2000-01), shows that soybean cultivation is mainly in 29 districts of 7 divisions: Nagpur, Kolhapur, Amarawati, Nasik, Pune, Aurangabad and Latoor (Table 3).

Table 3. Major Soybean-Producing Divisions and Districts of Three States of India. After the SOPA Report (2002-03).

Madhya Pradesh

Maharashtra

Division Bhopal

Districts Bhopal Sehore Raisen Vidisha Betul Raigarh Hosangabad/Harda

Division Nagpur

Districts Nagpur Wardha Chandrapur Bhandara Gadchiroli Gondia

Jabalpur

Jabalpur Katni Balaghat Chhindwara Mandla Dindori Seoni Narsinghpur

Kolhapur

Kolhapur Sangli Satara

Sagar

Sagar Damoh Panna Tikamgarh Chattarpur

Amrawati

Amrawati Yevatmal Akola Washim Buldhana

Rewa

Rewa Sidhi Satna Shadol Umaria

Nasik

Nasik Jalgaon Dhulia Nandurwar

Indore

Indore Dhar Jhabua Khargone Badwani Khandwa

Pune

Pune Ahmednagar Solapur

Rajasthan Districts Kota Bundi Baran Chittorgarh Udaipur Bhilwara Banswara Sawai Madhopur Jhalawar

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA

Ujjain

Ujjain Mandsour Neemuch Ratlam Dewas Shajapur

Aurangabad

Aurangabad Jalna Bid

Gwalior

Gwalior Shivpuri Guna Datia Murena Sheopur kalan Bhind

Latoor

Latoor Osmanabad Parbhani Hingoli Nanded

49

3.3. Rajasthan No soybean cultivation in Rajasthan was reported prior to 1952. The cultivation in Rajasthan was initiated in 1981-82 when 104 ha were planted (Agriculture in Rajasthan: Some facts, 2001). Both increases and decreases in the soybean cultivation area have occurred over the last five years. The area under cultivation in the years 1998-2003 varied as follows: 6.17 x 10 5 ha > 4.84 x 105 ha < 6.24 x 105 ha < 6.54 x 105 ha > 4.17 x 105 ha. Maximum yield (870 kg/ha) and production (5.4 x 105 tonnes) were recorded in 1998-99 and, in the subsequent years, a reduction in both yield and production was observed. The maximum decreases of 18.2% and 44.9% in the yield and production, respectively, were observed in 2002-03 as compared to 1998-99. Soybean cultivation spreads over nine districts of Rajasthan: Kota, Bundi, Baran, Jhalawar, Chittor, Udaipur, Bhilwara, Banswara, and Sawai Madhopur. Of these, Kota and Bundi are the main soybean-producing districts of Rajasthan (Table 3; SOPA Report, 2000-01). 4. REGIONAL DISTRIBUTION OF SOYBEAN CULTIVARS Under the soybean varietal development programme, intensive research has been conducted by various Universities and Research Institutes in India. These include: JNKVV, Jabalpur (Madhya Pradesh); National Research Center for Soybean (NRCS), Indore (Madhya Pradesh); GBP University of Agriculture and Technology, Pantnagar (Uttaranchal); Indian Council of Agricultural Research (ICAR), New Delhi; Indian Agricultural Research Institute (IARI), New Delhi; Punjab Agriculture University, Ludhiana (Punjab); Birsa Agriculture University, Ranchi (Bihar); Kalyani University, West Bengal; Gujarat Agriculture University, Gujarat, Punjab; Rao Agriculture University, Akola (Maharashtra); Banglore Agriculture University, Banglore (Karnataka); and Tamil Nadu Agriculture University, Coimbatore (Tamil Nadu). The yield, resistance towards pests and diseases, germinability, pod maturity time, improvement of germination, and lodging and shattering resistance were the major

50

MAHNA

targets. A good germplasm collection is maintained and evaluated at NRCS (Indore) to ensure their proper utilization in different zones of the country. From time to time over the last decade, improved varieties of soybean have been released for the different agro-climatic zones of the country. The released varieties consist of those of exotic as well as indigenous origin. From the point of view of agroclimatic conditions and varietal suitability, different regions of India are grouped into five soybean zones. Bhatnagar and Tiwari (1990), Ram (1996) and Bhatnagar (2002) have tabulated the varieties suitable for these five different soybean zones (Tables 4, 5 and 6). Table 4. Soybean Varieties Suitable for Different Zones of India. After Bhatnagar and Tiwari (1990).

S. No. 1

Zone

Area covered

Northern Hill Himachal Pradesh and hills zone of Uttar Pradesh

2

Northern Plain zone

Punjab, Haryana, Delhi, North eastern plains of Uttar Pradesh and Western Bihar Madhya Pradesh, Bhundelkhand region of Uttar Pradesh, Rajasthan, Gujarat and Northern Orissa

3

Central zone

4

Southern zone

Karnataka, Tamil Nadu, Andhra Pradesh, Kerala and Maharashtra

5

Northern Eastern zone

Assam, West Bengal, Bihar, Meghalaya, Sikkim, Arunachal Pradesh, Nagaland and Tripura

Suitable Varieties Bragg, NRC-2, PK-262, PK-308, PK-327, PK-416, Pusa-16, Pusa-20, Pusa-24, Shilajeet, Shivalik, VL Soya-1, VL Soya-2, VL Soya-21, VL Soya-47 and Hara Soya. Alankar, Ankur, Bragg, , PK-262, PK-308. PK-327, PK-416, PK-564, PK-1024, Pusa-16, Pusa-24, Shilajeet, SL-4 and SL-96. Bragg, Durga, Gaurav, Gujarat Soya1, Gujarat Soya-2, JS 80-21, JS 75-46, JS 71-05, JS-335, MACS-13, MACS57, MACS-58, Monetta, NRC-2, NRC-12, NRC-7, NRC-37, PK-472, PK 71-21, UPSM-19 and Alankar. Co-1, Hardee, KHSb-2, MACS-124, Monetta, PK-471, PK-1029, PK-472, Pusa-37, Pusa-40, MACS-450 and LSB-1. JS 80-21, Birsa Soya-1, Bragg, PK-472, Pusa-16, Pusa-22, Pusa-24 and MACS-124.

Description of 1979 germplasms in a published catalogue. Recommendation of RSC 2 and RSC 3 for cultivation due to their resistance towards stem borer pests. Enlistment of general characteristic features like germinability, resistance towards leaf shattering and various diseases of the four cultivars Ahilya-1 (NRC-2), Ahilya-2 (NRC-12), Ahilya-3 (NRC-7), Pooja (MAUS-2) and their suitability for cultivation in Himachal Pradesh, Madhya Pradesh, Uttar Pradesh, Rajasthan, Maharashtra, Gujarat. Recommendation of four rust resistant varieties namely PK-1024, PK-1029, JS-80-21 and Ankur for cultivation in disease affected area of Central India and Maharashtra, Karnataka, Tamil Nadu and Kerala. Data on an increase of 35% in the national yield in 1997-98 after adopting improved technology by farmers.

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA

51

Table 5. Soybean Varieties Suitable for Different Zones from 1996. After Ram (1996).

S. No 1

2

3

4

5

Zone Northern Hill zone

Northern Plain zone

Area covered

Varieties recommended

Himachal Pradesh and hills of Uttar Pradesh

Bragg, Lee, PK-262, PK-308, PK-327, PK-416, Pant Soybean-564, Pusa-16, Pusa-20, Shilajeet, Shivalik, VL Soybean-1 and VL Soybean-2 Alankar, Ankur, Bragg, Clark-63, PK-262, PK-308. PK-327, PK-416, Pant Soybean-564, Pusa-16, Pusa-22, Pusa-24, Shilajeet, SL-4, and SL-96 Bragg, Clark-63, Durga, Gaurav, Gujarat Soybean-1, Gujarat Soybean-2, JS-2, JS-80-21, JS-75-46, JS-76-205, MACS-13, MACS-58, Monetta, PK471, Pusa-16, Pusa-22 and T-49. Co-1, Davis, Hardee, Improved Palican, PHSb-2, Monetta, PK-471, Pusa-37 and Pusa-40

Punjab, Haryana, Delhi, North Eastern plains of Uttar Pradesh and Western Bihar Central zone M.P, Bundelkhand region of Uttar Pradesh, Orissa, Rajasthan, Gujarat, Northern and western parts of Maharashtra. Southern Karnataka, Tamil Nadu, zone Andhra Pradesh, Kerala and Southern parts of Maharashtra North-Eastern Assam, West Bengal, Bihar zone and Meghalaya

Birsa Soybean-1, Pusa-16, Pusa-22, Pusa-24, Alankar and Bragg

In the year 2000, the Rajasthan Seed Corporation Ltd., Jaipur, determined the maturity period, yield, and other important characteristic features of three cultivars, namely JS-335, Ahilya (NRC-12), and Ahilya-1 (NRC-2), which were recommended for cultivation in Rajasthan. Culitvars, NRC-7, JS-335, JS 71-05, L129 and NRC 25, were susceptible to insects. NRC 33, which exhibited field resistance, was recommended as a source for insect resistance (Annual Report, DARE/ICAR, 2000-01). Shrivastava and Shrivastava (2001) have listed soybean varieties that are recommended for ten agro-climatic regions of Madhya Pradesh (Table 7) and fifteen other varieties have been described in terms of maturity period, yield, and other important characteristics, suitable for cultivation in Madhya Pradesh (Table 8). Similarly, Khare et al. (2000) tabulated soybean varieties, which are important for various agroclimatic zones of Madhya Pradesh (Table 9). Seven soybean varieties, along with their important agronomical characters for various areas of adaptation (Madhya Pradesh, Rajasthan, Uttar Pradesh and Andra Pradesh), were described in 2001-02 (see Table 10). Khatri et al. (2002) have listed ten important pests along with the names of varieties showing resistance against specific pests that cause damage to soybeans (Table 11). Bhatnagar (2002) has tabulated 26 varieties of soybeans with their special characteristic features and duration of maturation (Table 12).

52

MAHNA Table 6. Soybean Varieties Suitable for Different Agro-climatic Zones of India from 2002. After Bhatnagar (2002).

S.No.

Zone

Area covered

Suitable varieties

1

Northern hill zone

Himachal Pradesh and hills of Uttar Pradesh

2

Northern plain zone

3

Central zone

4

Southern zone

Punjab, Haryana, Delhi, Eastern plain zones of Uttar Pradesh and Western Bihar Madhya Pradesh, Bundelkhand region of Uttar Pradesh, Rajasthan and North Eastern Maharashtra Karnataka, Tamil Nadu, Andhra Pradesh, Kerala, and Southern region of Maharashtra

5

North Eastern zone

Bragg, PK-262, PK-308, PK-327, PK-416, Pusa-16, Pusa-20, Pusa-24, Shilajeet, Shivalik, VL Soya-2 and NRC-2. Bragg, PK-262, PK-308, PK-327, PK-416, PK-564, Pusa-16, Pusa-24, Shilajeet, SL-2, SL-4, SL-96, PK-1024 and PK-1042. Bragg, JS 80-21, JS 75-46, JS 71-05, JS-335, Monetta, PK-472, Pusa-16, Pusa-24, Punjab-1, PK-416, NRC-2, NRC-12, NRC-7 and NRC-37. Co-1, Hardee, KHSB-2, KM-1, Monetta, PK-471, ADT-1, MACS-450, PK-472, MACS-58, PK-1029, MAUS-1, MAUS-2 and MACS-450. Birsa Soybean-1 and Bragg in Bihar hills, Alankar, Bragg, PK262, PK-472, Pusa-16, Pusa-24, and JS 80-21 in North-East region.

Assam, West Bengal, Bihar, Meghalaya, Chhattisgarh and Orissa

Table 7. Soybean Varieties Recommended for Different Agroclimatic Zones of Madhya Pradesh. After Shrivastava and Shrivastava (2001).

S. No.

Agroclimatic Zone

Varieties

1. 2.

Plains of *Chhattisgarh Kamour plateau and satpura hills

JS 72-280 (Durga), JS 80-21 and JS 335. JS 72-280, JS 72-44 (Gaurav), JS 75-46, JS 8021, JS 335, JS 90-41, MACS 58 and PK 472.

3.

Plateau of Vindhya

4.

Central Narmada valley

5. 6.

Gird region Bundelkhand region

7.

Satpura hills

8.

Malwa

9. 10.

Nimar valley Jhabua hills

Punjab-1, JS 72-44, JS 76-205, JS 80-21, PK-472. JS 72-44, JS 72-280, JS 75-46, JS 76-205, JS 80-21, JS 90-41 and PK-472. JS 80-21, JS-335. JS 72-44, JS 72-280, JS 75-46, JS 76-205, JS 80-21, JS-335, JS 90-41 and PK-472. Punjab-1, JS 75-46, JS 80-21, JS-335, JS 90-41, PK-472 and MACS-58. JS 72-44, Punjab-1, JS 71-05, JS 76-205, JS 80-21, JS-335, JS 90-4, PK-472, Ahilya-1, Ahilya-2, Ahilya-3. JS -335, JS 90-41. JS-335, JS 90-41.

* Chhattisgarh is separated from Madhya Pradesh as a new state.

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA

Table 8. Developed Varieties for Soybean Cultivation in Madhya Pradesh. After Shrivastava and Shrivastava (2001).

S. No.

Name of Variety

Maturation Period and Yield

Specific Properties

(A). Early maturing 1 2 3

Punjab–1 JS-71-05 JS-335

90-98 days, 1.8-2.0 t/ha 90-97 days, 2.0-2.4 t/ha 95-100 days, 2.5-3.0 t/ha

4 5

JS 90-41 NRC-7 (Ahilya-3) NRC-12 (Ahilya-2)

87-98 days, 2.5-3.0 t/ha 90-99 days, 2.5-3.5 t/ha

6

96-99 days, 2.5-3.0 t/ha

High seed germinability Resistant to pod dehiscence High seed germinability (80%) and high yield Four seeded pods Resistant to various bacterial and viral diseases Resistant to leaf blight and tolerant to YMV.

(B). Medium maturing 7

9

Gaurav (JS 72-44) Durga (JS 72280) MACS-13

10 11

JS 75-46 JS 76-205

12

PK 472

13

MACS-58

14

JS 80-81

104-106 days, 2.0-3.0 t/ha 106-108 days, 2.2-2.6 t/ha 106 days, 2.2-3.0 t/ha

15

NRC-2 (Ahilya-1)

103-106 days, 25-30 Qui/ha

8

106 days, 2.0-2.5 t/ha

102 days, 2.0-2.6 t/ha

Resistant to bacterial pustule and pod blight. High germinability (80%)

106-108 days, 2.0-2.6 t/ha 106 days, 2.0-3.0 t/ha 104 days, 2.0-2.5 t/ha

Resistant to YMV and high germinability (80%) Resistant to pod shattering. Highest germinability. Resistant to bacterial pustule, YMV and Rhizoctonia. Resistant to YMV and leaf spot. High germinability and resistant to leaf eating insect. Resistant to various fungal and bacterial diseases.

53

54

MAHNA Table 9. Soybean Notified Varieties Recommended for Different Agroclimatic Zones of Madhya Pradesh. After Khare et al. (2000).

S.No.

Agroclimatic Zone

Varieties

I.

Plains of Chhatisgarh

JS 72-280 (Durga), JS 80-21 and JS-335.

II.

Plateau of Baster

JS 80-21, JS–335

III.

JS 80-21, JS–335

V.

Northern hills of Chhatisgarh Kamour plateau and Satpura hills Plateau of Vindhya

VI.

Central Narmada valley

VII.

Gird region

VIII.

Bundelkhand region

IV.

JS 72-280, JS 72-44 (Gaurav), JS 75-46, JS 80-21, JS-335, JS 90-41, MACS 58 and PK-472. Punjab-1, JS 75-46, JS 76-205, JS 80-21, PK-472. JS 72-280, JS 75-46, JS 76-205, JS 80-21, JS-335, JS 90-41 and PK- 472. JS 80-21, JS-335.

IX.

Satpura hills

X.

Malwa

XI.

Nimar valley

JS 72-280, JS 75-46, JS 76-205, JS 80-21, JS-335, JS 90-41 and PK-472. Punjab-1, JS 75-46, JS 80-21, JS-335, JS 90-41, PK-472 and MACS-58. Punjab-1, JS71-05, JS 76-205, JS 80-21, JS-335, JS 90-4, PK- 472, Ahilya-1, Ahilya-2, Ahilya-3. JS-335, JS 90-41.

XII.

Jhabua hills

JS-335, JS 90-41.

Table 10. Soybean Varieties Notified in 2000-01. After Annual Report, DARE/ICAR (2000-01).

Variety

Area of Adaptation

Yield Potential (t/ha)

Remarks

Madhya Pradesh, Rajasthan, and Uttar Pradesh

1.8-2.0

Resistant to pod shattering and good germination, early maturity

A. Central Releases NRCS-37 (Ahilya-4)

55

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA

MAUS-47 (Parbhani Sona) Himso-1563 (Hara Soya)

Maharashtra, Madhya Pradesh, and Rajasthan Maharashtra, Madhya Pradesh, and Rajasthan

1.5-1.8

Culinary purpose Culinary purpose

1.5-2.0

B. State Releases

Indira Soya-9

Madhya Pradesh

1.5-1.8

MAUC-32

Maharashtra

1.8-2.0

Pant Soy 1092

Uttar Pradesh

_

LBS-1

Andhra Pradesh

1.2-1.5

Moderately resistant to rust, medium in maturity Medium duration Resistant to YMV, medium duration Medium duration

Table 11. Insect-resistant Varieties of Soybean. After Khatri et al. (2002).

S. No. 1

2

3

4 5

Common and Scientific Name of Insect Stem fly (Melanagromyza sojae)

Girdle beetle (Obereopsis brevis) Green Semi looper worm (Chrysodeixis acuta)

Brown Semi looper worm (Gisonia gema) Leaf borer insect (Billonmbata sabsesivella )

Resistant Varieties TAS 1-3-5-2, MAQS-14, SL-459, MACS-617, MACS-716, DSB-I, MACC-124, MACS-534, JS (SH) 90-91, JS 90-35, MAUM-47, JS (SH) 91-33, MAUS 92-3, CO-1, DS 93-39-2, RSC-2, RSC-3, TAS-41, SACS-428, JS-335, JS (SH) 88-86, MACS-380 and MACS-396 PK-1162, JS-335

NRC-34, JS (SH) 89-48, MAUS-33, DSB-1, RAC-1, RAC-2, NRC-3, JS 90-9, UGM-52, NRC-12 DSB-1, RCS- 1 and NRC-12, NRC-7, RAC-3, NRC-34 NRC-37, JS (HS) 89-48, MACS-124, MACS 92-47, SL-414, NRC-39, JS71-05, DS 93-352, UGM-52, JS-335, MAUS-610, MACS-716, TAS-41, MAUS-33, MAUS-31, JS 92-2, MACS-44

56

MAHNA

6 7 8 9

10

Leaf folding caterpillar (Hedylepta indica) Tobacco caterpillar (Spodoptera litura.) Bean stink bug (Challiopsis sp.) Mahu (Aphis sp.) White fly (Bemisia tabaci)

HRMSO-1564 MRMSO-1564, JS90-41, UGM-52, SL-459, DSK 93-204-101 NRC-36, NRC-39, BLS-2 JS-335, JS-7105, JS-88-66, SL-79, CO-2, PK-1092 PK-1241, JS-91-4, PK-1189, PK-1180, PK-1162, PK-1158, BRAG SL-317, SL452, SL-443, SL-450, PK-416, SL-427, PKB-25, SL-457, PK-1042, PK-1135.

Table 12. Improved Varieties of Soybean in India. After Bhatnagar (2002)

S. No.

Variety

Duration Day

Specific Features

1

Ahilya-1 (NRC-2)

103-106

2

Ahilya-2 (NRC-12)

96-99

3

Ahilya-3 (NRC-7)

90-99

4

Ahilya-4 (NRC-37)

96-101

Early maturity, White flower, Yellow seed, Good germination, Limited growth. Best for Malwa plateau and North hills region. Purple flower, Yellow seed, Brown hilum, Resistant to Bacterial pustule, Bacterial blight, YMV, GMV, Rhizoctonia aerial blight. Best for Bhundelkhund region. Limited growth, Purple flower, Yellow seed, Gray hair, Large seed, Early maturity, Resistant to pests and diseases. Best for Madhya Pradesh. White flower, Brown hairs, Yellow seed, Light dark brown hilum, Erect growth, Collar rot, Bacterial pustule resistant. Resistant to blight disease of pods and buds, Resistant to stem fly and Leaf minor.

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA 5

CO-2

75-80

6

Durga (JS 72-208)

102-105

7

JS 71-05

94-96

8

JS-335

98-102

9

JS-80-21

95-109

10

Sneha (KB-79)

85-93

11

MACKS 13

90-102

12

MACKS-58

95-105

Limited growth, Purple flower, deep leaves, yellow seed, Tolerant to YMV and Leafminor. Best for Tamil Nadu. White flower, Yellow seed, Early maturity and improve variety, Good germination, Resistant to bacterial pustule. Best for Kharif in M.P. and for Rabi in Chhattisgarh. Early maturity, Purple flower, Brown hair, Yellow seed, Black hilum, Dwarf determinate 35-40 cms height, Large seeds. Best for Malwa plateau of Madhya Pradesh. Purple flower, Yellow seed, Good germination, Semideterminate, Tolerance to pod dehiscence, Resistant to bacterial pustules, Blight or Alternaria blight. Tolerance to GMV, Stem fly, High yielding variety. Best for Bhundelkhund. Purple flower, Yellow seed, good germination, Tolerant to defoliators, Bacterial pustules. Best for central region, North East region and Chhattisgarh. Limited growth, Purple flower, gray hairs, Yellow seeds, Brown hilum, Tolerance to many diseases. Best for Karnataka. Purple flower, Yellow seeds, Good germination, tolerance to bacterial pustules, Viral disease and defoliators. Best for central region of India. Purple flower, Yellow seed, Tall plant, Tolerant to Bacterial pustule and leaf spot, YMV. Best for central region.

57

58

MAHNA 13

MACKS-124

90-105

Purple flower, Semi-Determinate. Best for southern region and Chhattisgarh.

14

MACKS-450

90-95

15

Pooja (MAUS-2)

102-110

16

PK-262

120-125

17

PK-416

100-109

18

PK-472

100-105

19

PK-564

105-115

20

PK-1024

115-118

21

PK-1029

90-95

22

PK-1042

110-119

23

PUSA-16

100-105 (Plains), 110-120 (Hills)

Purple flower, Medium height, Semi-Determinate, Yellow seed, Black hilum, Resistant to YMV and different diseases, Moderate resistant to stem fly and Insect pest. Best for southern region. Limited growth, Gray hair, Yellow seed, Brown hilum, Resistant to dehiscence of pods. Best for Maharashtra and Southern region. White flower, gray hair, Yellow seed, Brown hilum, Tolerant to YMV and Bacterial pustules. Best for Northern and North Plain region. White flower, Yellow seed, Resistant to Bacterial pustules and YMV, Tolerant to Rhizoctonia. Best for Northern region and Madhya Pradesh. White flower, Yellow seed, Deep green plant, Resistant to YMV, Leaf spot and Bacterial diseases. Best for Madhya Pradesh. Whit flower, Yellow seed, Deep brown flower hilum, Limited growth, Strong plant, Resistant to YMV and bacterial pustules, Tolerance to Rhizoctonia. Best for Northern region. White flower, Small leaves, Limited growth, Yellow seed, Brown hilum, Resistant to pod dehiscence, tolerant to rust disease. Best for Northern region. White flower, Limited growth, Broad green leaves, Yellow seeds, Black hilum and soybean rust disease. Best for Southern region. Limited growth, White flower, Yellow seed, and Brown hilum, Resistant to pods dehiscence. Best for Northern plain region. Purple flower, Yellow seed, Good germination, Resistant to Rhizoctonia, Bacterial pustules, YMV and Insect pests.

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA 24

Shivalik

120-125

25

VL Soya -2

104-116

26

VL Soya -21

120-122

59

White flower, Light milky hair, Yellow seed, Deep brown hilum, Resistant to middle height (90-100 cms) Yellow mosaic. Best for Himachal Pradesh. Purple flower, Light white hair, Black hilum, Light colour pods. Best for Northern hills. Limited growth, White flower, Brown hair, Yellow seed, Brown hilum, Good germination, Resistant to Bacterial pustules and Cercospora leaf spot.

5. REGIONAL AGRICULTURAL ASPECTS OF SOBEAN CULTIVATION Soybean, the main winter season crop of India, has wide adaptability to various agroclimatic conditions and can be fitted to all climatic systems (both rain-fed and irrigated). Production and productivity of soybeans sown in various agricultural regions of India depend basically upon rainfall, temperature, sowing, quality of soil, selection of suitable varieties for a specific agro-climatic region, control of diseases, fertilizer management, crop rotation, and intercropping. The following sections summarize and critically analyse the important agricultural aspects of soybean cultivation in India. 5.1. Environmental Factors During the soybean cultivation period (June-September), the situation with regards to the monsoon varies in different soybean-growing states of the country. Good rains occur up to the middle of July in most parts of the soybean belt. Rains may appear in the first week of August and may continue until the third week of August. Scattered rains also occur in September. Rainfall generally seizes after the second week of September, which is a very important period for pod filling and development. Most of the soybean cultivation in India depends upon rainfall. In the rain-fed areas, maximum cultivation is completed in two phases, i.e., early sown phase (between the second and third week of June) and timely sown phase (third week of June to the second week of July). As an exception, sometimes early sowing has been reported in the second week of May, as was done in Maharashtra in 2001-02. Timely sowing of soybean, which is accomplished by the arrival of the monsoon at the right time, provides optimum soil moisture for seed germination. Early sowing of the crop invariably gives a good yield, as reported in Maharashtra in winter 2000-01 (SOPA Report, 2001-02). The late arrival of the monsoon means both less rainfall and late sowing, which leads to the formation of undersized grains and less yield. Scattered rains in September can improve crop prospects (SOPA Report, 2000-01). Assessment of rainfall amounts for the recent years, 2000-2002, for the three main soybean-growing states of India, Madhya Pradesh, Maharashtra, and Rajasthan, indicates the rainfall

60

MAHNA

range at Division/District headquarters as well as the average state rainfall (Table 13). While discussing the rainfall status and requirement for the cultivation of soybean in different regions of Madhya Pradesh, Mehta and Shrivastava (2002) have pointed out that the cultivation is done in North-west Madhya Pradesh with the average rainfall of ca. 800 mm and in South-east Madhya Pradesh with the average rainfall of 600 mm. It is worthwhile to note that the amount of rainfall varies in each monsoon season and, as a consequence, all of normal, excess, deficient and scanty rainfall have been recorded in different years for different regions (SOPA Report, 2001-02). Table 13. Rainfall Status in Madhya Pradesh and Maharashtrain 2002, and Rajasthan in 2000. After SOPA Report (2002-03) and Agriculture in Rajasthan: Some Facts (2001).

Madyapradesh Divisions

Rainfall Range (mm)

Average (mm)

Jabalpur Sagar Rewa Indore Ujjain Gwalior Bhopal State Avg.

723-1456 662-868 435-1060 505-998 291-645 168-609 566-1318 479-993

942 700 690 670 659 417 844 703

Maharashtra Nagpur Kolhapur Amaravati Nasik Pune Aurangabad Latoor State Avg.

782-1223 250-1458 600-1293 504-898 366-601 572-758 443-1106 502-1048

917 862 894 694 445 639 744 742

Rajasthan - Rainfall from June 2000 to September2000 Av. Rain fall (Normal; mm) Districts Kota Bundi Baran Jhalawar Chittor Udaipur Bhilwara Banswara Swai Madhopur State Avg.

698 726 822 801 792 596 642 903 828 531

Av. Rain fall (Actual; mm) 585 529 814 638 486 391 440 545 475 365

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA

61

The optimum air temperature for seed germination is 30°C (20°C soil temperature) and temperatures above 40°C stop germination (Shrivastava et al., 2002). A temperature range of 25°C-28°C is found to be suitable for soybean growth. Even an increase of 2°C from the threshold temperature (29°C) causes a reduction in yield (Burke and Evett, 1999). 5.2. Quality of the Cropping Field Soil Cultivation of soybean is done in the well-drained, black loamy soil with a pH between 7.0-7.5 (Bhatnagar, 2002). Very light, sandy, acidic and alkaline soils are not suitable for this crop. In Madhya Pradesh, the soya state of the country, soybean is being cultivated in different soil types, such as shallow-black to dark-black soil (Malwa), medium-black (Vindhyan plateau), dark-black (Central Narmada valley), light-black loamy and silty loam (Satpura plateau), sandy loam to heavy-black (Kamour plateau), red-black mixed (Bhundelkhund), mixed red-black and alluvial (Gird region), and shallow-black (Nimar valley) (Shrivastava and Shrivastava, 2001; Mehta and Shrivastava, 2002). Seeds are sown in the soil at a depth of 2.5-3.0 cm, at a distance of 10-15 cm, and in rows maintaining 45 cm distance between two adjacent rows (Shrivastava et al., 2002). 5.3. Insect Pests, Diseases, and their Control Measures One of the reasons for the reduced soybean productivity is the increase in diseases caused by various organisms. Various researchers have provided an account of important diseases of soybean and their control measures. Singh et al. (1988) detailed the insect pests of soybeans in India, and then Singh and Singh (1989) classified and described the soybean pests and their control measures. The names and symptoms of the important diseases caused by fungi (seed rot, leaf spot, blight, root and stem diseases), bacteria (soybean rust, bacterial spot), and viruses (YMV), along with their control measures, have been reported by Khare et al. (2000). Pests that can spoil the cultivation of soybean due to their attack on different plant parts at different stages of growth have been described (Khatri et al., 2002; Shrivastava et al., 2002; see Table 14). In addition to insect-pest diseases, Khatri et al. (2002) also pointed out six main diseases of soybean caused by viruses (Yellow Mosaic Virus), fungi (Macrofomina sp., Cercospora sp., Colletotrichum slycence, Rhizoctonia solani, Focopsora pachyrhiza) and bacteria (Xanthomonas phaseoli var. sojans). Shrivastava et al. (2002), while listing the breeding objectives for soybeans, have pointed out that, with the increase of soybean-cultivation area, diseases are also spreading. Furthermore, out of forty diseases reported to occur in different soybean-growing regions of the country, seven fungal, two viral, eighteen pest, and one bacterial disease have been highlighted by them as being of economic importance. Biological and chemical treatments are recommended for the control of insects, fungal, bacterial, and viral diseases. The biopesticide (Bacillus bassiana) was found effective in controlling major diseases caused by insects (Annual Report,

62

MAHNA

DARE/ICAR, 1999-00). Khatri et al. (2002) have reported that the use of Nuclear Polyhedrosis Virus (NPV) reduces the number of tobacco caterpillars when sprayed with water. Table 14. Major Insect Pests of Soybean in India. After Khatri et al. (2002) and Shrivastava et al. (2002).

Common Name

Scientific Name

Damaged plant Parts & Symptoms

A. Seedling insects Blue beetle Field cricket Seed maggot Cut worms

Cneorane sp. Gryllus sp. Delia platura Agrotis ipsilon

Cotyledonary leaves Seed and seedling Seedling root Cotyledonary leaves Seed and seedling

B. Stem borer pests Stem fly Girdle beetle

Melanagromyza sojae Obereopsis brevis

Yellow stem, stunted growth of leaves Hollow stem, stem loss on maturation

C. Foliage feeder insects Green semi looper Brown striped semi looper Leaf minor caterpillar Tobacco caterpillar Linseed caterpillar Bihar hairy caterpillar Red hairy caterpillar Gram pod borer Looper larva Leaf folding caterpillar D. Sap suckers White fly Green jassids Thrips

Chrysodeixis acuta Mocis undata Aproaerema modicella Spodoptera litura Spodoptera exigua Spilosoma obliqua Amsacta moorei Helicoverpa armigera Scopula remotataguence Hedylepta indica

Chlorophyll loss, low yield Cut leaf margins Reduced growth Cut leaf margins Stuck leaves, cause holes Chlorophyll loss, low yield Reduced growth, leaf loss Leaf deformation 60-80% leaf loss, cause holes Cut leaf margins Leaf loss

Bemisia tabaci Empoasca terminalis Caliothrips indicus

Green stink bug Brown bug

Nezara viridula Riptortus pedestris

Cause Yellow leaves Shrunk pods Spread yellow mosaic viral disease Yellow leaves Shrunk pods

From time to time, various chemicals have been recommended for the control of soybean diseases. For pest diseases, these include Forate, Quinolphos, Aldosulfon, Methylparathion, Thymathaxam 70 (WS), Chloropyrifos 20 EC, or Ethian 50 EC; for fungal diseases, they include Indofill M 45, Bloytox 50, Hexaconezol 5 EC, or Propeconezol 5 EC; and for viral diseases, Mancojeb or Thiram + Carbendazim have been recommended (Annual Report, DARE/ICAR, 1997-98; Khare et al., 2000; Shrivastava and Shrivastava, 2001; Bhatnagar, 2002; Khatri et al., 2002 ).

AGRICULTURAL ASPECTS OF SOYBEAN IN INDIA

63

5.4. Soybean Weeds and their Control Measures Weeds and their management during soybean cultivation is a major problem and a cause of productivity loss. Weed species infesting soybean vary according to the agro-climatic region. The main weed species associated with soybeans, as reported by various researchers, are Cyperus rotundus, Echinochloa colonum, Digitaria sanguinalis, Phyllanthus maderaspatensis, Acalypha indica, Anotis montholoni, Dactyloctenium aegyptium, Commelina benghalensis, Saccharum spontaneum, and Phyllanthus niruri (Lokras et al., 1987; Sharma et al., 1991; Singh et al., 1991; Prabhakar et al., 1992; Upadhyaya et al., 1995; Jain et al., 1998). Mehta and Shrivastava (2002) have found that weeds not only reduce production but also cause the spread of various insect pests. Hand weeding has been adopted by farmers as the most effective control measure in the soybean-cultivated fields. Weed control is performed manually after a period of 20-25 days (Kurchania and Bhalla, 1999; Shrivastava and Shrivastava, 2001; Mehta and Shrivastava, 2002). Vyas et al. (1999) reported that the application of Alachlor as a pre-emergence measure resulted in better weed control and higher grain yield. Further, both Imazethapyr and Propaquizafop have shown promise for controlling weeds after their emergence (Annual Report, DARE/ICAR, 2000). Khare et al. (2000) and Shrivastava and Shrivastava (2001) have listed for soybean the herbicides to be applied before sowing (Fluchoralin, Metachlore), after sowing but before germination (Alachlore, Pendymethylin, Acetachlore, Metachlore), and on a standing crop (Imazethapyr). 5.5. Fertilizers Cow dung and compost fertilizer are used for maintenance of agricultural land for soybean cultivation. Khare et al. (2000) pointed out that soil characteristics are maintained, if 10-15 tonnes/ha compost is applied every third year. In general, nitrogen (20 kg/ha), phosphate (60-80 kg/ha), and potash (20 kg/ha) is applied at the time of sowing (Shrivastava and Shrivastava, 2001; Mehta and Shrivatava, 2002; Bhatnagar, 2002). Shrivastava and Shrivastava (2001) suggested that leguminous crops require higher sulfur concentrations compared to cereal crops. Therefore, sulfur-containing fertilizers, like ammonium sulfate, super phosphate, and gypsum, are used for soybean cultivation. Mehta and Shrivastava (2002) have noted that the addition of zinc sulfate to zinc-deficient soil and gypsum to sulfur-deficient soil is necessary to maintain soybean productivity. 5.6. Intercropping and Crop Rotation By adopting intercropping in the farming of soybeans, higher production can be achieved as compared to other crops. Patra and Chatterjee (1986), Prasad and Shrivatava (1991), and Jagtap et al. (1993) reported higher yields and return under soybean-based intercropping systems than with soybean alone. Crop rotation also helps in weed control (Kurchania and Bhalla, 1999). Soybean intercropping provides

64

MAHNA

safe production under unfavorable climatic conditions (Mehta and Shrivastava, 2002). Important intercropping combinations that are beneficial for different soybeangrowing regions of Madhya Pradesh have been recommended by Mehta and Shrivastava (2002), with the Soybean-plus-Arhar (Cajanus cajan) intercropping system found to be most beneficial in terms of production. Either soybean-plus-wheat or soybean-plus-chickpea are the major crop rotations, although other crops are also planted after soybeans (Shrivastava et al., 2002; see Table 15). In different agro-climatic regions, sowing of wheat or chickpea or pea or sunflower after soybean cultivation can result in much higher production, but the benefit depends on irrigation facilities (Mehta and Shrivastava, 2002). Although Chui and Shibles (1884) noted reduced yields of soybeans after intercropping with maize, Shrivastava et al. (2002) indicated that intercropping of soybean with maize (Zea mays), as well as with pigeonpea (Cajanus cajan), jowar (Sorghum vulgare), fingermillet (Elusine corocana), or sugarcane (Saccharum officinarum), is beneficial. Goswami (2000) also reported that the soybean-plus-maize intercropping system is productive. Table 15. Major Intercropping Systems involving Soybean in India. Region

Intercropping System

Malwa plateau

Soybean-plus-Maize (Zea mays) (4:2) or Soybean-plus-Arhar (Cajanus cajan) (4:2)

Kamour plateau

Soybean-plus-Arhar (Cajanus cajan) (4:2)

Vindhyan Plateau

Soybean-plus-Jowar (Sorghum vulgare)(4:2)

ACKNOWLEDGEMENTS The author thanks the European Union for support of the INCO DEV project “Soybean BNF Mycorrhization for Production in South Asia”, ICA4-CT-2001-10057. REFERENCES Agricultural statistics at a glance (2001). New Delhi: Department of Agricultural Statistics, Government of India. Agriculture in Rajasthan: Some facts (2001). Pant Krishi Bhawan, Jaipur, India: Statistical Cell Directorate of Agriculture. Annual Report (1997 – 1998). (pp. 57-59). New Delhi, India: DARE Ministry of Agriculture, Government of India, ICAR. Annual Report (1999 – 2000). (pp. 27-28). New Delhi, India: DARE Ministry of Agriculture, Government of India, ICAR. Annual Report (2000 – 2001). (pp. 30-31). New Delhi, India: DARE Ministry of Agriculture, Government of India, ICAR. Anonymous (1956). The wealth of India, Vol, IV. F-G. Raw materials (pp. 142-150). New Delhi, India: Council for Scientific and Industrial Research.

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Bhatnagar, P. S. (2002). How to earn more profit in soybean cultivation? Few easy methods and improved varieties of soybean Prasar. The Soybean Processors Association of India (SOPA), Indore, India. Bulletin 1, 1–22. Bhatnagar, P. S., and Tiwari, S. P. (1990). Soybean varieties of India. NRCS Technical Bulletin 2, 1-11. Burke, J. J., and Evett, S. R. (1999). Identification of the temperature threshold for soybean irrigation. In H. E. Kauffman (Ed.), Proceedings of the World Soybean Research Conference VI, (pp. 603). Chicago, IL: University of Illinois. Chui, J., and Shibles, R. (1984). Influence of spatial arrangement of maize on performance of an associated soybean intercrop. Field Crop Res., 8, 187–198. Goswami, D. (2000). Effect of crop geometry and nutrient management on soybean + maize intercropping system. Jawaharlal Nehru Krishi Vishna Vidhyalaya, Jabalpur, India. M.Sc. (Ag) Thesis. Hooker, J. D. (1879). Flora of British India, Vol. II. London, UK: L. Reeve and Co. Hymowitz, T., and Kaizuma, N. (1981). Soybean seed protein electrophoresis profiles from 15 Asian countries or regions: Hypothesis on path of dissemination of soybean from China. Econ. Bot., 35, 10– 23. Jagtap, J. G., Gupta, R. K., and Holkar, S. (1993). Evaluation of soybean and pigeonpea varieties for advantage in intercropping on the basis of stability analysis. Indian J. Agric. Sci., 63, 327-332. Jain, K. K. Parorkar, N. R., and Agrawal, K. K. (1998). Major weed flora of soybean field at Powarkheda (MP). Soybean Abstracts, 21, 166. Kale, F. S. (1936). Soybean, its value in dietetics, cultivation and use. Baroda, India: Baroda State Press, and New Delhi, India: International Book and Periodicals Supply Service. Kaltenbach, D., and Legros, J. (1936). Soya selection, classification of varieties, varieties cultivated in various countries. Institutes of Agriculture (Rome, Italy). Monthly Bulletin of Science and Practical Agriculture 27, 284. Khare, D., Rawat, N. D., Bhaley, M. S., and Shrivatava, A. N. (2000). Soyabean seed production technology (pp. 1-22). Jawaharlal Nehru Krishi Vishna Vidhyalaya, Jabalpur, India: Seed Technology Unit Transmmision Center. *Khatri, A. K., Shukla, B. N., and Sharma, A. (2002). Soybean Phasal mein samanavit keet avum rog prabhandhan (Integrated pest and disease managements in soybean crops), (pp. 1-22). Jawaharlal Nehru Krishi Vishna Vidhyalaya, Jabalpur, India: Seed Technology Unit Transmmision Center. *Kurchania, S. P., and Bhalla, C. S. (1999). Kharif phasal mein neenda niyantran (Weed control in Kharif crops). Jawaharlal Nehru Krishi Vishna Vidhyalaya, Jabalpur, India: Department of Agronomy, Technical Bulletin 1. Lokras, V. G., Singh, V. K., and Tiwari, J. P. (1987). Chemical weed control in soybean (Glycine max (L.) Merrill). Indian J. Weed Sci., 17, 45-48. *Mehta, S. K., and Shrivastava, V. C. (2002). Soybean ki unnat kasht (Improved cropping of soybean). (pp. 1-16). Jawaharlal Nehru Krishi Vishna Vidhyalaya, Jabalpur, India: Department of Plant Breeding and Genetics. Paroda, R. S. (1999). Status of soybean research and development in India. In H. E. Kauffman (Ed.), Proceedings of the World Soybean Research Conference VI, (pp. 13-23). Chicago, IL: University of Illinois. Patra, A. P., and Chatterjee, B. N. (1986). Intercropping of soybean with rice, maize and pigeonpea in the plains of West Bengal. Indian J. Agric. Sci., 56, 413-417. Prabhakar, Tiwari, S. P., and Bhatnagar, P. S. (1992). Production technology for augmenting soybean productivity in India. J. Oilseeds Res., 9, 1-13. Prasad, K., and Shrivastava, V. C. (1991). Pigeonpea (Cajanus cajan) and soybean (Glycine max) under intercropping system under rainfed situation. Indian J. Agric. Sci., 61, 243-246. Ram, H. H. (1996) Soybean breeding. In J. Nizam, S. A. Farook, and I. A. Khan (Eds.), Genetic improvement of oilseed crops, (pp. 27-42). Hyderabad, India: Ukaaz Publications. Saxena, M. C., Pandey, R. K., and Hymowitz, T. (1971). Agronomic requirement of soybean (Glycine max (L.)Merr). Indian J. Agric. Sci., 41, 339-344. Saxena, M. C., and Pandey, R. K. (1971). Characterstics and performance of some promising varieties of soybean at Pantnagar. Indian J. Agric. Sci., 41, 355-360 Sharma, R. K., Bangar, K. S., Kanere, G., Singh, O. P., Thakur, G. L., and Sharma, S. R. (1991). Effect of weed control on yield of soybean (Glycine max (L.) Merrill). Indian J. Agron., 37, 372-373.

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*Shrivastava, A. N., and Shrivastava, M. K. (2001). Soybean ki unnat kheti (Improved farming of soybean). Transmission Center, Jawaharlal Nehru Krishi Vishna Vidhyalaya, Jabalpur, India. Krishi Vishva Kharif Special, 29, 52-63. Shrivastava, A. N., Singh, C. B., and Shrivastava, M. K. (2002). Soybean. In C. B. Singh and D. Khare (Eds.), Genetic improvement of field crops, (pp. 136-157). Jodhpur, India: Scientific Publishers. *Singh, A., Pandey, S., and Sharma, Y. K. (2001). Swasthayavardhak soya aahar (Health improving soya food). Jawaharlal Nehru Krishi Vishna Vidhyalaya, Jabalpur, India: Department of Food Science and Technology Transmission Center. Singh, B. B., and Saxena, M. C. (1975). Soybean varieties for different agro-climatic zones in India. Seed Technology News, 5, 5-7. Singh, O. P., and Singh, K. G. (1989). Insect pest control in soybean. In O. P. Singh and S. K. Shirivastava (Eds.), Soybean, (pp.113-132). Bikaner, India: Agro Botanical Publishers India. Singh, O. P., Verma, S. N., and Nema, K. K. (1988). Insect pests of soybean. Dehradun, India: India International Book Distributors and Publishers. Singh, V. K., Bajpai, R. P., Mishra, R. K., and Purohit, K. K. (1991). Chemical weed control in rainfed soybean (Glycine max (L.) Merrill). Indian J. Agron., 36, 292-294. SOPA Report (2000-01). A report on soybean crop estimates, (pp. 1-8). Indore, India: The Soybean Processor Association of India. SOPA Report (2001-02). A report on soybean crop estimates, (pp. 1-6). Indore, India: The Soybean Processor Association of India. SOPA Report (2002-03). A report on soybean crop estimates, (pp. 1-4). Indore, India: The Soybean Processor Association of India. Tiwari, S. P., Bhatnagar, P. S., and Prabhakar (1994). Identification of suitable soybean (Glycine max) varieties for non-traditional regions of India. Indian J. Agric. Sci., 64, 872-874. Tiwari, S. P., Joshi, O. P., and Sharma, A. N. (1999). The advent and renaissance of soybean in India - The saga of success, (pp. 1-58). Indore, India: National Research Council for Soybean (ICAR). Upadhayay, V. B., Tiwari, J. P., and Kosta, L. D. (1995). Influence of weed control methods on soybean (Glycine max (L.) Merrill) yield. JNKVV Research Journal, 27, 1-4. Vyas, M. D., Singh, S., and Singh, P. P. (1999). Efficacy of the herbicide alachlor in soybean. Soybean Abstracts, 22, 89. Williams, R. (1932). Flora of India or Description of Indian plants, 2 nd Ed., Vol. II, (pp. 134-315). Calcutta, India. * Original in Hindi

Chapter 5

SOYBEAN CULTIVATION AND BNF IN CHINA J. E. RUIZ SAINZ1, J. C. ZHOU2, D.-N. RODRIGUEZ-NAVARRO3, J. M. VINARDELL1 and J. E. THOMAS-OATES4 1

Department of Microbiology, Faculty of Biology, University of Sevilla, Apdo-1095, 41080-Sevilla, Spain. 2 Department of Microbiology, Huazhong Agricultural University, Shi Zi Shan Street, P.O. Box 430070, Wuhan, People’s Republic of China. 3 Centro de Investigación y Formación Agraria “Las Torres y Tomejil”, Apartado Oficial 41200-Alcalá del Río, Sevilla, Spain. 4 Department of Chemistry, University of York, Heslington, York, YO10 5DD, U.K.

1. SUMMARY China is the geographical origin of cultured soybeans and is where they have been cultivated for more than 5,000 years. Soybean is grown in nearly all provinces of China, the exceptions being Qinghai and the Tibetan Plateau. Based on climatic differences, China can be divided into five main geographical regions as regards soybean cultivation. As a complementary classification, Chinese soybean varieties have also been classified according to the sowing date; they were divided into winter-, spring-, summer-, and autumn-sowing varieties. In spite of the soybean acreage in China (9.4 million ha in 2001), which ranks fourth in the world, China has also become the largest soybean importer in the world. Although China has greatly improved soybean productivity in the last 20 years, its total production (or average seed yield) is still clearly lower than that reached in western countries. The symbiotic interaction, which soybean forms with different rhizobia, is a key factor in increasing soybean productivity in China within the context of agricultural sustainability. Research efforts in China have mainly concentrated on the identification and characterisation of biological material, both soybean accessions and rhizobial strains that effectively nodulate soybeans. As a result of this activity, a large collection of soybean germplasm is available and the existence of a broad spectrum of bacteria that nodulate soybeans (which we will collectively call “soybean-rhizobia”) has been demonstrated. However, although soybean 67 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 67-87. © 2005 Springer. Printed in the Netherlands.

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inoculation is increasing as a soybean-cropping practice in China, the available scientific information on the results obtained in field experiments is negligible. Consequently, this review will be mainly focused on a description of: (i) the Chinese geographical areas devoted to soybean cultivation; (ii) the diversity of soybean-rhizobia and their populations in Chinese soils; and (iii) the collection of soybean germplasm available in China. Information about crop rotation (soybean with non-legumes) and the undesirable effects that frequently accompany continuous soybean cropping is also provided. 2. SOYBEAN CULTIVATION IN CHINA: HISTORICAL ASPECTS AND CURRENT SITUATION 2.1. Historical records and world-wide dissemination of soybean cultivation China is the geographical origin of cultured soybeans and the use of this crop in agricultural practices is more than 5,000 years old. The first written record is found in the book, “Pen Ts’ao Kong Mu”, in which the plants of China are described (2838 B.C.). Further, in similar ancient writings, Zou Zhuan said that “one of Zhou Zi’s brothers was so foolish that he could not tell Shu (an ancient name for soybean) from wheat”. Soybean was also one of the five sacred grains (or “Wu Ku”), that include rice, soybean, wheat, barley, and millet. In Hubei province, soybean ashes have been found in a Han tomb (second century B.C.). Although it is clear that the soybeans used in agriculture originated from wild soybeans, it is not clear in which location soybean was cultivated for the first time. It may be that the Yellow River valley was the first place ever at which soybean was cultivated. Although soybean is now grown in many countries, its expansion outside Asia only took place during the last three centuries. 2500 years ago, soybean cultivation was exported to what is now Korea and, 500 years later, to the islands we now call Japan. By the 7th century A.D., soybeans were grown in all southern countries neighbouring China. In 1712, the German botanist Kaempfer brought soybeans to Europe. Soybean was exhibited in the Botanical Garden of Paris in 1739 and in the British Royal Botanic Gardens in 1790. In 1804, the United States of America started to grow soybeans as a forage crop rather than for harvesting for seed consumption. Soybean cropping was initiated in other countries in Europe (18751877) as a result of its successful exhibition at an International Exposition in Vienna in 1873. Although soybean was not cropped in Brazil until the 1960s, this country is at present the second largest soybean producer after the United States of America. Soybean cropping was spread through Africa during the twentieth century. 2.2. Current soybean production in China At the present time, China ranks third internationally in soybean acreage and production. In spite of this position, however, the increasing domestic demand for

SOYBEAN AND BNF IN CHINA

69

soybean forced China to start soybean importation in 1995. Since then, Chinese imports of soybean have increased dramatically, with China at present the largest soybean importer in the world. China represents the largest market for U.S.A. soybean exports, which were valued at more than $1 billion in 2001. The other main sources of soybean imports into China are Argentina and Brazil. Soybean is grown in nearly all provinces of China with the exception of Qinghai and the Tibetan Plateau (see Figure 1 and Table 1). The number of soybean varieties that are available in China is very large; the Chinese Soybean Germplasm collection contains more than 23,000 soybean accessions. This large soybean diversity is the result of cultivation over such a large territory, with wide variations in topography and climate, and a long history of soybean cultivation. Chinese soybean varieties have been divided according to the season of the year in which they are sown. Most of the soybean varieties fall into one of the main three categories: spring-, summer- or autumn-sowing soybeans. There are some winter varieties that can be used in the south of China (in Hainan, Yunnan, Guangxi, and Guangdong provinces).

Figure 1. The Provinces of China.

70

RUIZ SAINZ, ZHOU, RODRIGUEZ-NAVARRO, VINARDELL, AND THOMAS-OATES Table 1. Weather Conditions, Soybean Acreage, and Soybean Production in the Different Regions of China.

Province

Annual rainfall (mm)

Shanghai Xinjiang

1000 150

Jiangsu

7801170 Shandong 550850 Jilin 400950 Sichuan 4001300 Zhejiang 9001400 Beijing 550660 Hunan 12001700 Henan 6001200 Hubei 7501500 Fujian 10002000 Guang1350dong 2600 Jiangxi 12001900 Liaoning 4851130 Heilong400jiang 600 Gansu 35-810 Hebei 400800 Yunnan 6002300 Anhui 7701700

Total Annual Soybean Total annual average acreage soybean sunshine temperature (ha x 103) production (h) (ºC) (tons x 104) 1531 15-16 6.2 1.9 2550-3550 North: -4/9 62.8 17.5 South: 7-14 1980-2640 ----249.2 67.0

Average yield (kg/ha)

3065 2787

2689

2335-2768

12-14

458.2

104.6

2283

1000-2900

-----

539.0

120.3

2232

1100-2700

-1/20

169.6

37.4

2205

1800-2300

15.2-18.2

129.0

28.4

2201

2084-2873

4-12

22.1

4.7

2127

1300-1800

16-18

205.8

42.8

2080

2000-2400

12-16

564.7

115.8

2051

1600-2200

13-18

224.8

45.8

2037

1670-2405

14.5-21.3

105.4

20.5

1945

1450-2300

18-24

96.9

18.7

1930

1473-2077

16-20

152.5

25.9

1689

2285-2870

4.8-10.4

301.9

48.1

1593

2400-2800

0-4

2868.3

450.1

1569

1700-3300 2500-3100

0-15 4-13

88.2 423.7

13.4 62.9

1519 1485

910-2800

4.9-23.7

52.0

7.7

1481

1730-2500

14-16

682.2

91.5

1341

SOYBEAN AND BNF IN CHINA Shanxi

Guangxi

Guizhou

Tianjin Inner Mongolia Shaanxi

Ningxia

Tibet

350----700 1100- 1500-1800 2800 1100- 1200-1600 1300 900 2204 50-450 -----

400- 1288-2885 900 178- 2195-3082 680 North: 1600-3400 10 ºC): 1900-4000 ºC. Annual rainfall: 350-1200 mm. Soybean-cultivation period: April-September.

72

RUIZ SAINZ, ZHOU, RODRIGUEZ-NAVARRO, VINARDELL, AND THOMAS-OATES Loess plateau spring soybean subregion: North Yangtze River, North Shanxi and Shaanxi provinces, InnerMongolia Plateau, irrigating area of the Great Bend of Huanghe River and Ningxia province Northwest spring soybean sub-region: Xinjiang and scattered zones in Gansu corridor

Non-frost period: 260-280 days. Activity accumulated temperature (>10 ºC): 3000-3500 ºC. Annual rainfall: 200-250 mm. Soybean-cultivation period: April-September.

Non-frost period: 110-200 days. Activity accumulated temperature (>10 ºC): 1500-3000 ºC. Annual rainfall: 300-500 mm. Soybean-cultivation period: April-September. II. HuangheNon-frost period: 175-220 days. Middle Hebei and Shanxi Huaihe-Haihe Activity accumulated subregion: parts of Hebei River basin temperature (>10 ºC): province south to the Great 3800-4300 ºC. Wall but north to Shijiazhuang Annual rainfall: 400-800 mm. and Tianjin cities, and middle Soybean-cultivation period: and Southeast parts of Shanxi June-September. province. Huanghe-Huaihe-Haihe River Non-frost period: 180-220 days. Activity accumulated basin subregion: the part of temperature (>10 ºC): Hebei province south to 4000-4800. Shijiazhuang and Tianjin City, Shandong province, most parts Annual rainfall: 500-1000 mm. Soybean-cultivation period: of Henan province, Hongze June-September. district of Jiangsu province, those parts of Anhui province north to Huaihe River, the south western parts and Guangzhong district of Shanxi province and Tianshui district of Gansu province. Non-frost period: 210-310 days. III. Spring and Yangtze basin spring and Activity accumulated summer sub-region: Yangtze summer temperature (>10 ºC): River basin in Jiangsu, Anhui regions of 4500-5500 ºC. Yangtze River and Sichuan provinces, Hubei Annual rainfall: 1000-1500 mm. province, southern parts of basin Soybean-cultivation period: Henan and Shaanxi province, April-July or May-September. northern parts of Zhejiang province and Sichuan basin.

SOYBEAN AND BNF IN CHINA Yunnan plateau spring soybean subregion: parts of Hunan, Sichuan and Guangxi provinces

IV. Southeast spring and autumn soybean region

Southern parts of Zhejiang province and most parts of Fujian, Taiwan, Hunan, Guangdong, and Guangxi provinces

V. South China Southern edge of Guangdong, Yunnan and Guangxi four-season provinces, some parts of soybean region Fujian, and Hainan province.

73

Non-frost period: All year. Activity accumulated temperature (>10 ºC): 5000-6000 ºC. Annual rainfall: 750-1000 mm. Soybean-cultivation period: April-July or May-September. Non-frost period: 270-320 days. Activity accumulated temperature (>10 ºC): 5500-7500 ºC. Annual rainfall: 1000-2000 mm. Soybean cultivation period: April-July or July-November Non-frost period: All year. Activity accumulated temperature (>10 ºC): 7500-9000 ºC. Annual rainfall: 1300-2000 mm. Spring soybean: February-June. Summer soybean: June-August. Autumn soybean: JulySeptember. Winter soybean: DecemberApril.

1

The information in this Table is gathered from the book “Breeding and Cultivation of Soybean in China” (1987, edited by the Academy of Agricultural Sciences of Jilin province, China) and also from The Atlas of Chinese Climate Resource (1994, China Meteorological Administration, China Map Press, Beijing China).

Soybean acreage and productivity in China has been increased in the last 20 years. In 1981, soybean acreage was 8 million ha, giving a total soybean production of 9.3 million tons. These figures demonstrate that Chinese soybean productivity was low because the national average yield was only 1163 kg/ha. Results presented in Table 3 clearly show that China has made great efforts in increasing the productivity of soybean cultivation. In comparison to 1981, in 2000, soybean acreage was 9.3 million ha that gave a total production of 15 million tons. This increase in total soybean production is mainly attributable to higher yields (1656 kg/ha) rather than to an increase in soybean acreage. In spite of the improvements in farming practices in combination with improved soybean cultivars, soybean productivity in China is still clearly lower that reached in Western countries (information on world-wide soybean production they can be obtained from different web address, such as http://www.fas.usda.gov/psd/complete tables/OIL-table224.htm). In 2001, soybean acreage in the U.S.A. was ca. 29.5 million ha, which gave a total production of 78.6 million tons (an average seed yield of 2660 kg/ha).

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In the same year, the soybean acreage in China was about 9.4 million ha, which rendered a total production of 15.4 million tons (an average seed yield of 1630 k/ha). In that same year, the average seed yield in the other two main soybeanproducing countries (Argentina and Brazil) was 2120 and 2660 kg/ha, respectively. These results clearly show that the main goal for increasing soybean production in China is to increase seed yield. Table 3. Soybean Production in China in the last Two Decades. This table was constructed by using the information published in China Agriculture Yearbook, 2001, China Agricultural Press, Beijing, China (in Chinese).

Years

1981-1985 1986-1990 1991-1995 1996-2000

Soybean acreage (ha x 103) 7.801 8.090 (3%) 8.212 (5%) 8.317 (6%)

Total soybean production (tons x 104) 966 1.134 (17%) 1.302 (34%) 1.455 (50%)

Average yield (kg/ha) 1.238 1.402 (13%) 1.585 (28%) 1.749 (41%)

Numbers in brackets refer to increases over the period 1981-1985.

Table 1 also shows the average seed yield of each Chinese province. Shanghai and Xinjiang provinces show the highest yields, but the soybean acreage in these two provinces is very small, so that it does not represent the general situation in China. Jiangsu province is the only one that, having a large soybean acreage (249,000 ha), produces a seed yield that is in line of those obtained by the U.S.A. and Brazil. Most of the other provinces showing seed yields over 2000 kg/ha are located either on the east coast of China (Jilin, Shandong, Jiangsu and Zhejiang) or on a North-South axis in Central China (Henan, Hubei and Hunan), with Sichuan province (Central-West China) being the exception. Neighbouring some of the most productive provinces, there are others (such as Liaoning or Anhui) that have a large soybean acreage but their productivity is clearly much lower. The province with the largest soybean acreage, Heilongjiang, is situated in the North of China and although its seed yield (1569 kg/ha) is clearly lower than that in the above mentioned provinces, it is comparable with that obtained in other cold regions, such as Canada (1520 kg/ha in 2001). It is clear that the main constrains for increasing soybean productivity in China are of a different nature (such as weather conditions, soil fertility, water availability, farming technology, etc.) in the different soybean-cropping areas. Increasing soybean productivity through symbiotic nitrogen fixation is a clear option. However to our knowledge, although soybean inoculation is gaining popularity among Chinese farmers, very little scientific information about the symbiotic performance of soybean inoculants under field conditions is available. Due to this lack of information, we focus our attention on the biological resources (soybeanrhizobia and the soybean germplasm collection) that exist in China and on a description of the soybean-rhizobia populations that are indigenous to soybeancropping areas.

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3. NITROGEN-FIXING BACTERIA THAT NODULATE SOYBEAN 3.1 Diversity of soybean-nodulating rhizobia in China Because China is the geographical origin of soybeans and they have been cropped in China for at least the last five millenia, it is reasonable to expect that China is the appropriate place for carrying out screenings aimed at the isolation of new soybeannodulating rhizobia strains. This idea is based on the hypothesis that co-evolution between the two symbionts (the plant and the bacteria) is likely to have taken place and, consequently, that it would be possible to isolate new soybean-nodulating rhizobia species and also bacterial strains showing improved symbiotic performance with soybeans. Taxonomic studies carried out in the last 20 years have shown that bacteria belonging to different genera and species are able to nodulate soybeans. Until 1982, soybeans were believed to be nodulated only by slow-growing bacteria belonging to the species, Bradyrhizobium japonicum. Since then, however, new groups of soybean rhizobia have been isolated and classified. Soybean-nodulating bacteria are distributed among different species belonging to three different genera: (i) Bradyrhizobium japonicum, B. elkanii and B. liaoningense, which are slowgrowing bacteria with generation times longer than six hours (van Berkum et al., 1998); (ii) Mesorhizobium tianshanense, with a variable generation time (Tan et al., 1997); and (iii) Sinorhizobium fredii and S. xinjiangense, which are fast-growers with generation times of 1.5 and 4 hours, respectively (Chen et al., 1988). The slow-growing strains belonging to the genus Bradyrhizobium produce alkali in a medium containing mannitol. Those Bradyrhizobium strains able to nodulate soybeans have been classified into 17 different serogroups and also into three different DNA homology groups (for a review Rhizobiaceae taxonomy, see van Berkum and Eardly, 1998 and volume 3 of this iseries). DNA-homology groups I and Ia appear to be closely related and separated from DNA-homology group II. These differences, as well as differences in other bacterial traits, such as EPS composition, fatty acids, or intrinsic antibiotic-resistance patterns, led to the segregation of those strains belonging to DNA-homology group II into the new species called B. elkanii. In these studies, slow-growers isolated from Asia were underrepresented. Strains belonging to Bradyrhizobium liaoningense were isolated from several different Chinese provinces, including Liaoning, Heilonjinag, or Hubei (Xu et al. 1995). Their doubling time is so long (ranging from 19 to 39 hours) that they are commonly referred as extra-slowly growing (ESG) soybean rhizobia. Some strains are highly effective with the Chinese soybean cultivar Heinong 26. B. liaoningense poorly nodulated Phaseolus aureus and failed to nodulate both Lotus sp. and Astragalus sinicus. The level of DNA homology between EGS strains and either B. japonicum or B. elkanii is lower than 40% (Xu et al., 1995). Mesorhizobium tianshanense strains (originally called Rhizobium tianshanense) were isolated from an arid saline desert soil of Xinjiang, the most westerly province of China (Chen et al., 1995). The strains comprising the species M. tianshanense were isolated from nodules of different legumes, such as Glycyrrhiza padilliflora, G. uralensis, Sophora alopecuroides, or Glycine max. M. tianshanense strains

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produced acid in a medium containing mannitol and their generation times varied from 5-15 hours. Those isolates from soybean nodules, strains 009B and 91X01, are slow growers and show a generation time of 10 and 13.5 hours, respectively. Partial 16S rRNA gene sequencing of the type strain CCBAU3306 revealed that this strain is closely related to M. loti, but not to B. japonicum (Chen et al., 1995). Fast-growing soybean bacteria were first isolated 20 years ago (Keyser et al., 1982) and were finally clustered into the species, Sinorhizobium fredii (Chen et al., 1988). These first soybean isolates are able to form nitrogen-fixing nodules on Asiatic soybean cultivars (such as cultivar Peking) but either fail to nodulate or are very poorly effective with the modern cultivars from North America (Keyser et al., 1982; Buendía-Clavería and Ruiz-Sainz, 1985; Buendía-Clavería et al., 1989). Later, new S. fredii strains, which are able to form nitrogen-fixing nodules with both Asiatic and American soybean cultivars, were isolated (Dowdle and Bohlool, 1985; Yang et al., 2001; Camacho et al., 2002). In addition to their marked soybean cultivar specificity, S. fredii strains show a very broad host range, being able to nodulate at least 79 different genera of legumes (Pueppke and Broughton, 1999). Most of the well-studied strains of S. fredii are from China (Keyser et al., 1982; Dowdle and Bohlool, 1985; Yang et al., 2001; Camacho et al., 2002; RodríguezNavarro et al., 2002), although fast-growing soybean microsymbionts have also been isolated from Malasia (Young et al., 1988), Vietnam (Cleyet-Marel, 1987) and Panama (Henández and Focht, 1984). Taxonomic studies have shown that S. fredii is closely related to the alfalfa microsymbiont, Sinorhizobium meliloti (de Lajudie et al., 1994), although both their host ranges and nodulation-factor structures (Nod factors or LCOs) are different (Dénarié et al., 1996; Gil-Serrano et al., 1997). They are also related to Sinorhizobium teranga and S. saheli (de Lajudie et al., 1994), two species isolated from nodules of West African trees. The species Sinorhizobium xinjiangense also clusters with fast-growing bacteria that form nitrogen-fixing nodules on soybeans and other legumes, such as Vigna unguiculata and Cajanus cajan (Chen et al., 1988). It was isolated from soil and soybean nodules collected in Xinjiang province. S. xinjiangense strains can be differentiated from those belonging to S. fredii by a number of physiological characteristics, such as carbon and nitrogen utilisation, intrinsic antibiotic resistance patterns, or the ability to grow at acid or alkaline pH. 3.2. Studies of the natural soybean-rhizobia populations in Chinese soybean cropping areas Considering the extensive soybean-cropping areas in China, China’s different types of soils and its climatic diversity, there are surprisingly few reports on the indigenous soybean-rhizobia populations, their symbiotic nitrogen-fixation capacity, and their competitive ability to nodulate soybeans (Dowdle and Bohlool, 1985; 1987; Chen et al., 1988; Buendía-Clavería et al., 1994; Xu et al., 1995; Li et al., 1996; Li Fu-Di, 1996; Jiang et al., 1996). All these facts are of relevance to the ultimate goal of producing rhizobial inoculants to increase soybean productivity

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under field conditions. To reduce this gap in information was one of the main objectives of a project, which was funded by the European Union (1997-2000) and involved research institutions from China, Great Britain, the Netherlands, and Spain. In this project, indigenous fast- and slow-growing soybean rhizobia populations (most probably Bradyrhizobium japonicum and Sinorhizobium fredii, respectively) in soil samples from Shandong, Henan, Hubei, and Xinjiang autonomous regions were quantitatively analysed using the Most Probable Number (MPN) technique (Yang et al., 2001). Different Asiatic and American soybean cultivars were used as the trapping host. Table 4 shows that all soil samples contained large indigenous soybean-rhizobia populations, which were a mixture of fast- and slow-growing soybean rhizobia. Table 4. Estimation by the MPN Technique of the Soybean-Rhizobial Populations in Soil Samples from Shandong, Henan, Hubei and Xinjiang Autonomous regions. After Yang et al., 2001

Province of origin of the soil sample 1 Henan (7.5)

Xinjiang (7.3)

Hubei (8.0)

Shandong (7.5)

Soybean cultivar2

Estimated number of soybean-rhizobia 3

Heinong 33 Linzhen Williams Bragg Jing Dou 19 Heinong 33 Williams Kobe Jing Dou 19 Williams Heinong 33 Lizhen Williams Bragg

4 x 104 - 5 x 105 ----2 x 104 - 3 x 105 ----4 x 104 - 5 x 105 6 x 102 - 8 x 103 -----

104 – 105 104 – 105 9 x 104- 106 ----2 x 103 - 3 x 104 -----

Ratio fast- / slow-growers (%) 4 73/27 a 71/29 a 65/35 a 68/32 a 100/0 99/1 8/92 7/93 81/19 90/10 27/73 a 32/68 a 29/71 a 36/64 a

1 The pH of each soil sample is indicated in brackets. 2Heinong 33, Linzhen, and Jing Dou 19 are Chinese soybean cultivars, whereas Williams, Bragg, and Kobe are American soybean cultivars. 3The number of soybean-rhizobia in each soil sample was estimated in soybean plants that were grown at the pH of the soil sample. 4Statistical analyses are among cultivars inoculated with the same soil sample. For each soil sample, numbers on the same column followed by the same letter are not significantly different at the 5% level, using the Fisher test for comparing proportions. Statistical analyses were not carried out for the Xinjiang sample because the numbers are very different.

Apparently, fast-growers are more abundant than slow-growers in the Henan and Hubei soil samples, whereas in Shandong soil, slow-growers appear to be more abundant. Xinjiang soil is composed of soybean-rhizobia populations showing marked soybean-cultivar specificity because the ratio of fast-growers to slowgrowers isolated from soybean nodules varies with the soybean cultivar used as the trapping host. With Asiatic cultivars, most of the nodule isolates are fast-growers,

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whereas with American soybean cultivars, most of the isolates are slow-growers. Hence, the size of the indigenous rhizobial populations can be seriously underestimated if only one cultivar is used to determine the bacterial population. This cultivar specificity exhibited by soybean-rhizobia strains not only discriminates between Asiatic and American soybean cultivars but also among Asiatic cultivars (Jiang et al., 1996; Yang et al., 2001), which it is not surprising when the large diversity of soybean cultivars available in China is considered. Several thousand isolated soybean rhizobial strains are stored at the Department of Microbiology of the University of Seville in Spain and at the Institute of Biology of the University of Leiden in The Netherlands. Although only a fraction of the collection has received attention so far (Yang et al., 2001; Camacho et al., 2002; Thomas-Oates et al., 2003), the physiological, genetic and symbiotic characteristics a set of 198 fast-growing soybean-nodulating rhizobial strains from the four Chinese regions mentioned above have been studied (Thomas-Oates et al., 2003) and the results compiled in the first extensive catalogue of fast-growing soybean rhizobia, which is available at http://www.soybeanrhizobia.net. Table 5 summarizes the results from the study of some of the bacterial traits. In a new EU-funded project, an enlarged consortium is evaluating the indigenous population of soybean-rhizobia in soil samples from another 11 provinces that cover an axis from the North to the South of China. Soil samples are from both traditional soybean-cropping regions and also from areas where soybean might be introduced for the first time if the plans of the Chinese Government to increase soybean acreage are implemented. The emerging picture from the MPN experiments indicates that most of the soils contain high levels of soybean-rhizobia populations (in the range of 104 bacteria/g of soil), although some locations (for instance, some counties in Chongqin and Inner Mongolia), in which soybean is not cropped, appear to have lower levels (about 102 rhizobia/g of soil) of indigenous soybean-rhizobia (our unpublished results). In Heilongjiang, the Chinese province with the largest soybean acreage, the level of soybean-rhizobia populations is extremely high (107 bacteria/g of soil). These results are in accord with those previously reported by Jiang et al. (1996). They describe Hunan, Shanxi and Liaoning soybean-cropping areas as containing high levels of soybean-rhizobia (ca. 104/g of soil), whereas a soil sample from a Shandong field, which was not being used for soybean cultivation, had a lower (ca. 102/g of soil) soybean-rhizobia content. 3.3. Soybean inoculation Production of rhizobial inoculants and their application as an agricultural practice started in China in the 1950s. The first inoculants produced were for peanut and soybean. In the 1960s and 1970s, inoculants (Mesorhizobium huakii) for Astragalus sinicus were widely applied to increase rice production in a rotation system. A. sinicus is a traditional Chinese green manure, which is grown in winter and dug into

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the soil the next spring. Field experiments in Hubei, Henan, Anhui and Jiangxi provinces, where soils contained low levels of indigenous M. huakii populations, showed significant crop increases. Table 5. Physiological, Genetic and Symbiotic Properties of Fast-growing Soybean Rhizobia isolated from Soil Samples from Soybean-cropping Areas of Xinjiang, Henan, Shan Dong and Hubei Provinces

Bacterial trait

Remarks

Number of plasmids

All strains contain from 1-6 plasmids. A few strains contain very small plasmids whose size is in line with those previously reported.1 Acidification of medium Only 5% of the isolates did not show a significant containing mannitol acidification of the medium Growth at different pH Some strains grew in the pH range 5.5-9.0 Growth at 37ºC Very rare; only 2% of the strains analysed. Melanin production Very common; in 57% of the analysed strains. Generation time Most (75%) had generation times of 1.5-3.5 hours. A majority of the strains showed an LPS profile which Lipopolysaccharide is in line with those already reported for other S. fredii (LPS) profiles on strains2. polyacrylamide gels PCR finger-printing Some differences in the RFLP of 16S rDNA or 16S-23S analyses3 rDNA IGS PCR-amplified fragments. RAPD analyses also showed differences among strains Symbiotic Most are able to form nitrogen-fixing nodules with both characteristics American and Asiatic soybean cultivars. Marked bacterial-strain/soybean-cultivar specificity was found among Asiatic soybean cultivars. Some strains are highly effective with Asiatic soybean cultivars, but very few fix N2 with the American soybean cultivar Williams at a level comparable to that B. japonicum USDA110. 1

In a previous study carried out with a different set of soybean-rhizobia strains, very small indigenous plasmids were found. Strains CH12 and CH154 have indigenous plasmids of about 7.5 and 22 kb, respectively (Camacho et al. 2002). 2As described by Reuhs et al. 1999: A region (LPS-II) consisting of fast-migrating electrophoretic bands (Rough-LPS) that usually are not well resolved and another region (LPS-I) containing slow-migrating few slow-migrating bands (Smooth-LPS). 3RFLP, Restriction Fragment Length Polymorphism; IGS, Intergenic Spacer; RAPD, Random Amplified Polymorphic DNA.

In the 1980s, soybean-rhizobia inoculants were used on a large scale in China (on ca. 650,000 ha). In field conditions, the use of soybean inoculants has to contend with the problem that most of the soils investigated contain high populations of indigenous soybean rhizobia (Jiang et al., 1996; Yang et al., 2001). In these circumstances, the percentage of nodules occupied by the inoculant strains can be too low to achieve a significant contribution to the total fixed-nitrogen obtained through symbiotic nitrogen fixation (for a review of the competition problem, see Robleto et al., 2000). This problem is particularly serious if the

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natural rhizobial populations are poor nitrogen fixers, such as appears to be the case in Heilongjiang province, where as many as 22% of the growing soybean plants contain ineffective, or poorly effective, nodules (Li, 1996). Indigenous soybeanrhizobia strains, which also show mediocre nitrogen-fixation capacity, occur in the soils of other provinces (such as Henan, Hubei or Shanxi) (Jiang et al., 1996). Furthermore, soybean inoculation, as a method to increase soybean yield, failed in several experiments conducted in different parts of China, such as the Yellow River valley (Jiang et al., 1996). Positive responses to inoculation have, however, been reported. For instance, a high percentage of nodule occupancy by the B. japonicum strains used as inoculants was observed in a field experiment carried out at the farm of Huazhong Agricultural University (Wuhan, Hubei province), if the inoculant was incorporated into the soil rather than on the surface of the soybean seed (Li, 1996). Because this soil contained indigenous soybean-rhizobia populations (ca. 4 x 104 bacteria/g of soil), this strategy has the potential to increase the number of nodules formed by the inoculant. Increases of 11% in soybean seed yield have also been reported in field experiments in Heilongjiang, Sichuan and Guangxi, using a genetically engineered B. japonicum HN32 inoculant that contains a DNA fragment from S. fredii (Zhang et al., 1996). Table 6 shows the increases in soybean seed yield obtained by inoculation with strain HN32 in yearly field experiments conducted during the period 1990-1996. Although the increases are not dramatically high, they are consistent over all the field assays and over a very large acreage. Other soybeaninoculation experiments carried out in Heilongjiang, Jilin, and Liaoning provinces showed increases in soybean yield that varied from 6-33%. Table 6. Increases in Soybean Seed Yield in Field Experiments using B. japonicum HN32 as Inoculant.

Year

Province

Number of locations used

1990 1991 1992 1993 1994 1995 1995 1996 Total in 1990-1996

Heilongjiang Heilongjiang Heilongjiang Heilongjiang Heilongjiang Heilongjiang Sichuan Guangxi

7 5 7 9 10 1 1 1 41

1

Acreage used in the field experiments 5.797 25.665 23.997 18.678 29.329 2.666 2.000 11.200 119.332

Yield increase (%) over the uninoculated control1 9.8 10.1 8.5 11.8 11.5 11.7 13.0 11.7 11.0

Numbers refer to mean values for the different locations investigated. Seed yields ranged from 1375 to 2900, most being over 2000 kg/ha.

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Regardless of the competition problem posed by the indigenous soybeanrhizobia populations, soybean inoculation is actively promoted in China. For instance, in 2000, a company operating in Harbin city (Heilongjiang Lufeng Biological Organic Fertilizer LTD) and belonging to the Heilongjiang Academy of Science inoculated 400 ha. Because the company claims that positive responses to inoculation were scored, the production of soybean inoculants has been scaled up by using 10,000 litre fermentors. To our knowledge, however, information on soybean-nodule occupancy by the applied inoculant is not available. As far as we know, experiments in China, using 15N isotopically labelled nitrogen fertiliser, have only been carried out at the greenhouse level. From these pot experiments using 15N, it was concluded that the percentage of fixed-nitrogen derived from air (Ndfa%) varied from 40% to 60% among the Chinese cultivars analysed. The symbiotic performance of B. japonicum strains with 20 different Asiatic soybean cultivars was better than that shown by S. fredii strains (Yuangsheng et al., 1995). 4. THE SOYBEAN GERMPLASM COLLECTION IN CHINA Because soybean is native to China, the soybean germplasm collection is very large. Reports providing information about this collection are already available on the Internet; the Chinese Academy of Agricultural Sciences (CAAS) provides information at its web address http//www.caas.com.cn. An article, entitled Evaluation of Soybean Germplasm in China (Zhanyou et al., CAAS, Beijing, China), can be found at the web address http://www.gsf99.uiuc.edu/invited/2-305.pdf. The information presented here is a summaryof the information presented on the web pages mentioned above as well as other documents available via the internet, such as that presented in http://www.gsf99.uiuc.edu/invited/2-3-07.pdf. The National Gene Bank at the Institute of Crop Germplasm Resources in CAAS, Beijing, contains 23,587 soybean accessions, including 3000 wild soybean accessions. Twenty-eight provinces (all Chinese provinces except Qinghai and Tianjing) have provided local soybean varieties to the germplasm collection. As mentioned before, Chinese soybean varieties are divided according to the sowing date, into winter, spring, summer and autumn soybean varieties. The collection has 11,206 spring-sowing accessions (47% of the total collection), 11,648 summersowing accessions (49%), 733 autumn-sowing accessions (3%), and some wintersowing accessions. A large proportion of the stored accessions (22,637 out 23,587) have been characterised using six different aspects: Morphological characteristics: these include growth habit, stem termination, and flower and pubescence colours. Many accessions produce stems that are erect (67.3% of the whole collection) and show a determinate termination (51.7%). The total number of accessions producing either white or purple flowers is similar (10,467 and 12,138, respectively). Seed characteristics: these include colour, shape and size of seeds. Most of the soybean accessions have yellow cotyledons (96.8%) and yellow seed coats (60.5%). The collection also has accessions showing other seed coat (green, black, brown, and bicolor) or cotyledon (green) colours. Although accessions having ellipse-

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shaped seeds are the most abundant (61.9%), representatives of other seed shapes (flat ellipse, flat round, long ellipse, and kidney) are also present. In general, seeds of Chinese soybeans are mostly small (32.6%) or medium (42.9%) in size. Accessions having larger size seeds are also present in the collection (including some with extremely large seeds that weigh >30g/100 seeds). Seed nutrient quality: the protein content of soybean accessions in the collection varies from 29.3% to 52.9%. Accessions containing 40-48% protein are the most abundant (81.9%). The variety, Yan Tian Qinq Pi Dou, has the highest protein content (52.9%). The fat content of most soybean accessions is below 20%. Tolerance to environmental stress: although the whole collection has not been evaluated for either drought or salt tolerance, 463 accessions proved to be drought tolerant and only nine accessions were salt tolerant at the seedling stage. Most of the accessions were not tolerant to cold conditions, with only nine accessions able to germinate at low temperatures. Resistance to diseases: especially to soybean cyst nematode (SCN), which is a major disease affecting this crop. Some of the soybean accessions showed high resistance, or even immunity, to some SCN races, whereas all accessions were susceptible to a range of other SCN races. None of the accessions investigated showed high tolerance, or immunity, to soybean rust and only 69 accessions were moderately resistant. Soybean frogeye leaf-spot (SFLS) is a very serious disease that leads to poor seed production quality. In a screening of 961 accessions, 14 of them showed high resistance to SFLS in leaves, stems and pods. Genetic diversity: DNA finger-printing techniques were applied to 90 soybean accessions. RAPD markers were useful tools to identify soybean germplasm resources. The largest diversity of soybean accessions in the collection was found among those collected from Shanxi province.

5. SOYBEAN IN CROP ROTATION AND IN CONTINUOUS CULTIVATION Legumes can contribute fixed-nitrogen to cropping systems in several ways. An input of fixed-nitrogen into the soil will occur if the total fixed-nitrogen in the plant residues left after harvesting is greater than the total amount of fixed-nitrogen absorbed from the soil. Among all legumes cultivated in China, soybean is that one most commonly cropped. Higher economic benefits and environmental conditions account for this preference. Crop rotation between soybean and other relevant nonlegume crops is also used in China to enrich the soil N status for the following nonlegume crop. In Jilin province, for instance, corn yield was 18% higher in fields where soybean had been previously cropped than in those where rice was previously grown. Similarly, rice and wheat yields increased (16% and 31%, respectively) if soybean was previously cultivated. The crop-rotation strategies involving soybean vary according to the Chinese geographical area. Table 7 indicates some of the soybean/non-legume combinations most commonly used. In addition of the well-known benefits to the non-legume crop, rotation of legume and other crops avoids the decrease in soybean yield that is observed if soybean is continuously cropped in the same area. Unfortunately, in some areas, in

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Table 7. The Use of Soybean in Crop-Rotation Practices in Different Regions of China. From Zhang Mengcheng, 1999. Problems and Solutions on Cultivation of Soybean High Yield. China Braille Press, Beijing, China. (In Chinese).

Region Northeast China

Valley of the Yellow and Huaihe River

Valley of the Yangze River

Southeast China

Crop-rotation strategy Remarks Soybean – corn – broomcorn – corn Only one crop per Soybean – broomcorn (or rice) – corn year. Spring soybean – spring wheat One crop per year Winter wheat – summer soybean – or three crops in winter wheat - summer soybean – corn two years. Winter wheat – summer soybean – no crop in winter – cotton Winter wheat – summer soybean – Two or three crops winter wheat – summer corn per year. Winter wheat – summer soybean – winter corn – cotton Winter wheat – summer soybean – winter wheat – rice Winter wheat – summer soybean and corn – winter wheat – sweet potato Water supply is Early rice – autumn soybean – low in autumn. winter wheat or corn Soybean is Early rice – autumn soybean – frequently sown green manure or fallow in Winter after the rice Spring soybean – late rice – harvest. winter wheat or corn

Corn, Zea mays; broomcorn, Sorghum bicolour; sweet potato, Ipomoea batatas; wheat, Triticum aestivum; rice, Oryza sativa.

which the soil is poorly drained, normal strategies for crop rotation cannot be applied, so that soybean appears as the only possibility to local farmers. Research projects, supported by the Chinese Academy of Sciences (in 1986), The Department of Science and Technology of Heilongjiang (in 1988), and The Ministry of Science and Technology of China (in 1995), have been carried out in order to understand the main reasons underlying soybean and soil deterioration through continuous soybean-cropping practices (for a review, see Liu and Herbert, 2002). Table 8 compiles some of the negative effects that have been observed when soybean is continuously cultivated. Different crop-management practices have been proposed as possible solutions to alleviate the decline of soybean productivity when rotation is not possible (Liu and Herbert, 2002). These include: the adoption of resistant or tolerant soybean cultivars; a better control of soybean diseases and pest insects (for instance, by seed coating with fungicides and/or insecticides); the correction of the imbalance of macro- and micronutrients in the soil; or the application of suitable tillage practices.

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RUIZ SAINZ, ZHOU, RODRIGUEZ-NAVARRO, VINARDELL, AND THOMAS-OATES Table 8. Negative Effects on Soybean Plants, Soil Microbial Communities, and Soil Characteristics as a Result of Continuous Soybean Cropping. After Liu and Herbert, 2002 (The publications listed in the table are in Chinese)

Type of effect

Description

References

Plant growth and development

Liu et al., 1990 Wang et al., 1995a Wang et al., 1995b Xu and Wang, 1995 Xu et al., 1999

Microbial communities of the rhizosphere.

Shorter stem; yellowing of leaves; reduction of root development and leaf area; poor canopy development; reduction of the number of nodules; reduction of root dry matter; increase of flower and pod shedding. Decrease of pod and seed number; decrease of seed size; increased number of diseased seeds by infection with fungi and virus; increase in seed protein content; decrease in seed oil Increase in praline and phenolic compounds; decrease of superoxide dismutase and catalase activities; decrease in chlorophyll a and b; decrease in root sugar content; decrease in P, K, Zn, Mo, and B content; increase in Ca absorption; imbalance in K/Ca ratio; decrease in lipid membrane plasticity. Increase in phytopathogenic fungi (such as Penicillium, Botrytis, Fusarium and Gliocladium), soybean cyst nematodes, and pest insects Decrease in bacterial populations; increase in fungi populations; decrease in total microbial populations.

Soil characteristics, enzymatic activities, and soil compounds

Acidification of soil; decrease in phosphatase, urease, invertase and dehydrogenase activities; increase in soil phenol content; decrease in polysaccharides and soil aggregates.

Seed quality and yield

Physiological and biochemical plant characteristics

Plant diseases

Liu and Yu, 2000 Xu et al., 1997 Xu et al., 1999 Yang, 1997 Zheng, 1999 Han and Xu, 1996 Han and Xu, 1997 Jia and Yu, 1995 Liu et al., 1997 Zhao et al., 1998 Zheng et al., 1995

Xu and Wang, 1995 Wang et al., 1989 Hu et al., 1996 Han and Xu, 1997 Yu et al., 1988 Liu et al., 1990 Xu and Wang, 1995 Jia and Yu, 1995 Fu and Yang, 1999 Jia and Yu, 1995

6. CONCLUSIONS Soybean production in China is key to improving living standards for many farmers. Although Chinese soybean acreage ranks fourth in the world, its productivity is clearly lower than that of western soybean-producing countries. Research efforts

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have been mainly directed at collecting and characterising soybean varieties and soybean-rhizobia strains that form nitrogen-fixing nodules with this legume. Most of the soybean-cropping areas of China contain high levels of indigenous soybeanrhizobia, which represent a constraint for the successful use of soybean inoculants. Information from field experiments in China using soybean inoculants is not abundant. In contrast to reports on the failure of the inoculants to increase soybean productivity, there are also some reports that indicate that soybean inoculants might be successfully used for such a purpose. Due to the strong cultivar specificity exhibited by soybean-rhizobia and the fact that large collections of bacterial strains and soybean varieties are available, it might be possible to identify soybean cultivars that, while highly effective with a particular set of soybean-rhizobial strains, are also poorly nodulated by the indigenous soybean-rhizobia populations of a particular soil. The quality of the inoculants used and the way they are applied are also important questions that need to be thoroughly evaluated if this sustainable option is to become preferred over chemical fertilisation. ACKNOWLEDGEMENTS Part of the work presented here was founded by EU grants ERBIC18CT970191 and ICA4-CT-2001-10056. REFERENCES Buendía-Clavería, A. M., and Ruiz-Sainz, J. E. (1985). Isolation of mutants of fast-growing soybean strains that are effective on commercial soybean cultivars. Physiol. Plant., 64, 507-512. Buendía-Clavería, A. M., Chamber, M. M., and Ruiz-Sainz, J. E. (1989). A comparative study of the physiological characteristics, plasmid content and symbiotic properties of different Rhizobium fredii strains. Syst. Appl. Microb., 12, 203-209. Buendía-Clavería, A. M., Rodríguez-Navarro, D. N., Santamaría-Linaza, C., Ruiz-Sainz, J. E., and Temprano Vera, F. (1994). Evaluation of the symbiotic properties of Rhizobium fredii in European soils. Syst. Appl. Microbiol., 17, 155-160. Camacho, M., Santamaría, C., Temprano, F., Rodríguez-Navarro, D. N., Daza, A., Espuny, R., Bellogín, R., Ollero, F. J., Lyra, M. C. C. P., Buendía-Clavería, A., Zhou, J., Li, F. D., Mateos, C., Velázquez, E., Vinardell, J. M., and Ruiz-Sainz, J. E. (2002). Soils of the Chinese Hubei province show a very high diversity of Sinorhizobium fredii strains. Syst. Appl. Microbiol., 12, 592-602. Chen, W. X., Yang, G. H., and Li, J. L. (1988). Numerical taxonomy study of fast-growing soybean rhizobia and a proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int. J. Syst. Bacteriol., 38, 392-397. Chen, W., Wang, E., Wang, S., Li, Y., Chen, X., and Li, Y. (1995). Characteristics of Rhizobium tianshanense sp. nov., a moderately and slow growing root nodule bacterium isolated from an arid saline environment in Xinjiang, People’s Republic of China. Int. J. Syst. Bacteriol., 45, 153-159. Cleyet-Marel, J. C. (1987). Dynamique des populations de Rhizobium et de Bradyrhizobium dans le sol et la rhizosphere. These d’Etat. University Claude Bernard, Lyon, France. Dénarié, J., Debellé, F., and Promé, J. C. (1996). Rhizobium lipo-quitooligosaccharide nodulation factors: Signalling molecules mediating recognition and morphogenesis. Annu. Rev. Biochem., 65, 503-535. De Lajudie, P., Willems, A., Pot, B., Dewettnick, D., Maestrojuan, G., Neyra, M., Collins, M. D. Dreyfus, B., Kersters, K., and Gillis, M. (1994). Polyphasic taxonomy of rhizobia: Emendation of the genus Sinorhizobium and description of Sinorhizobium meliloti comb. Nov., Sinorhizobium saheli sp. nov., and Sinorhizobium teranga sp. nov. Int. J. Syst. Bacteriol., 44, 715-733. Dowdle, S. F., and Bohlool, B. B. (1985). Predominance of fast-growing Rhizobium japonicum in a soybean field in the People’s Republic of China. Appl. Environ. Microbiol., 33, 990-995.

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Thomas-Oates, J., Bereszczak. Edwards, E., Gill, A, Noreen, S., Zhou, J. C., Chen, M. Z., Miao, L. H., Xie, F. L., Yang, J. K., Zhou, Q., Yang, S. S., Li, X. H., Wang, L., Spaink, H. P., Schlaman H. R. M., Harteveld, M., Díaz, C. L., van Brussel, A. A. N., Camacho, M., Rodríguez-Navarro, D. N., Snatamaría, C., Temprano, F., Acebes, J. M., Bellogín, R. A., Buendía-Clavería, A. M., Cubo, M. T., Espuny, M. R., Gil, A. M., Gutiérrez, R., Hidalgo, A., López-Baena, F. J., Madinabeitia, N., Medina, C., Ollero, F. J., Vinardell, J. M., and Ruiz-Sainz, J. E. (2003). A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China. Syst. Appl. Microbiol., (in revision). Van Berkum, P., and Eardly, D. (1998). Molecular evolutionary systematics of the Rhizobiaceae. In H.P. Spaink, A. Kondorosi, and P.J.J. Hooykaas (pp. 1-24). The Rhizobiaceae, Molecular Biology of Model Plant-associated Bacteria. Dordrecht, The Netherlands: Kluwer Academic Press. Wang, Z. Y., Wang, Y. X., and Chen, T. L. (1989). Preliminary studies of continuous soybean on growth and development. Soybean Science, 10, 31-36. Wang, G. H., Xu, Y. L., and Liu, X. B. (1995a). Root exudates in relation to continuous soybean barriers. Soybean Science, 14, 158-161. Wang, G. H., Xu, Y. L., and Jiang, X. Y. (1995b). Plant residuals in relation to continuous soybean barriers. Systematics Sciences of Comprehensive Studies in Agriculture, 4, 8-13. Xu, L. M., Ge, C., Cui, Z., Li, J., and Fan, H. (1995). Bradyrhizobium liaoningense sp. nov., isolated from the root nodules of soybeans. Int. J. Syst. Bacteriol., 45, 706-711. Xu, Y. L., and Wang, G. H. (1995). Yield comparison of sterilized CS soil with control. Heilongjiang Agricultural Science, 6, 8-12. Xu, Y. H., He, Z. H., and Liu, Z. T. (1997). Effect of continuous soybean on seed protein and oil content. Soybean Science, 16, 319-327. Xu, Y. L., Liu, X. B., and Han, X. Z. (1999). Effect of continuous soybean on growth and development and yield. Sci. Agric. Sinica, 32 (Suppl.), 64-68. Yang, S.S., Bellogín, R.A., Buendía, A., Camacho, M., Chen, M., Cubo, T., Daza, A., Díaz, C.L., Espuny, M.R., Gutiérrez, R., Harteveld, M., Li, X.H., Lyra, M.C.C.P., Madinabeitia, N., Medina, C., Miao, L., Ollero, F.J., Olsthoorn, M.M.A., Rodríguez, D.N., Santamaría, C., Schlaman, H.R.M., Spaink, H.P:, Temprano, F., Thomas-Oates, J.E., van Brussel, A.A.N., Vinardell, J.M., Xie, F., Yang, J., Zhang, H.Y., Zhen, J., Zhou, J., and Ruiz-Sainz, J.E. (2001). Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations. J. Biotechnol., 91, 243-255. Yang, X. J. (1997). Screening of tolerant soybean cultivars (lines) to continuous soybean on biomass of soil microorganism. Soybean Science, 15, 357-361. Young, C. C., Chang, J. Y., and Chao, C. C. (1988). Physiological and symbiotic characteristics of Rhizobium fredii isolated from subtropical-tropical soils. Biol. Fertil. Soils, 5, 350-354. Yu., G. R., Liu, J. X., and Su, G. Y. (1988). Number of fungi and bacteria in topsoils of continuous soybean and sunflower system. Chin. J. Appl. Ecol., 7, 1-8. Yuansheng, M., Xuanjun, F., and Min, L. (1995). Biochemistry and Genetics of Nitrogen Fixation. Beijing, China: Chinese Science and Technology Press (in Chinese). Zhang, X. X., Zhou, J. C., Ma, L. X. Zhang, Z. M., and Chen H. K. (1996). Cloning and sequencing of a 3.7 kb enhancing fragment from Rhizobium fredii B52. In F. D. Li, T. A. Lie, W. X. Chen and J. C. Zhou (Eds.). Diversity and Taxonomy of Rhizobia (pp. 255-262). Beijing, China: China Agricultural Scientech Press.

Chapter 6 SOIL STRESS FACTORS INFLUENCING SYMBIOTIC NITROGEN FIXATION M. J. SADOWSKY Department of Soil, Water, and Climate, and The BioTechnology Institute, University of Minnesota, St. Paul, MN 55108, U.S.A.

1. INTRODUCTION The soil environment is under a constant state of change and, as such, can be relatively stressful for both macro- and microorganisms. Fluctuations in pH, nutrient availability, temperature, and water status, among other factors, greatly influence the growth, survival, and metabolic activity of soil microorganisms and plants, and their ability to enter into symbiotic interactions. Despite this situation, soils represent one of Earth’s most productive ecospheres, accounting for a majority of primary and successional productivity. Consequently, microbes, plants, and other soil inhabitants have evolved to adapt to the ever changing and often inhospitable soil environment. In this Chapter, I will discuss stress factors in soils that influence symbiotic nitrogen fixation and I will do so from the perspective of both the host plant and the microsymbiont. However, the reader should be aware that, whereas some stress factors simultaneously affect both symbiotic partners, e.g., water stress, others may differentially influence each partner to a seemingly different degree by different mechanisms. Moreover, both plants and microbes have often adopted different strategies for dealing with these stress factors. 2. IMPORTANCE OF SYMBIOTIC NITROGEN FIXATION Nitrogen (N) is one of the major limiting nutrients for most crop and other plant species (Newbould, 1989). Moreover, fixed-N acquisition and assimilation is second in importance only to photosynthesis for plant growth (Vance, 1998; Graham and Vance, 2000). From several indications and by many estimations, the 89 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 89-112. © 2005 Springer. Printed in the Netherlands.

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world will no doubt face severe food shortages in the not-too-distant future, in part due to excessive population growth and the negative environmental impact associated with this increased growth. Although Waggoner (1994) suggested that the Earth's population will reach nearly twice its current level of over 10 billion people by the year 2035, there is still some debate on when this will actually occur. However, there is little debate that this will indeed occur sometime in the not-toodistant future. In addition, populations in developing (and less developed) countries, which reside in the tropical and subtropical regions of Asia, Africa, and Latin America, may account for ca. 90% of the projected world population. Today, in tropical countries, plant materials provide ca. 80% of the caloric and dietary protein needs of individuals and this situation is not expected to change in the near future. In the fairly recent past, humans used ca. 10% of the total fixed carbon that is produced by plants through photosynthetic activity (Golley et al.,, 1992); today, humans use ca. 40% of that carbon. Moreover, it is estimated that, by 2030, humans will require ca. 80% of all photosynthetically-fixed carbon to meet their dietary requirements. Taking it as a given that enhanced agricultural production will require the utilization of large areas of land that are now considered to be either marginally productive or even non-arable, several alternate strategies are needed to meet these considerable and increasing human dietary needs in the future. Many diverse biological associations contribute to N2 fixation (BNF) in both soil and aquatic systems (Sprent, 1984). However, in most agricultural systems, the primary source of biologically-fixed N (ca. 80%) occurs via the symbiotic interactions of legumes and soil bacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Allorhizobium, Mesorhizobium, and Azorhizobium (Sadowsky and Graham, 1998; Vance, 1998). The other 20% is contributed mainly by the actinorrhizal (e.g., by Frankia) and Anabena-Azolla types of symbiotic interactions. It has been estimated that legumes provide approximately 35% of the worldwide protein intake and that ca. 250 million ha of legumes are currently grown world wide. These symbiotic partners together fix an astounding 90 Tg N per year (Kinzig and Socolow, 1994) which points to the obvious conclusion that enhancing the both the use and management of biologically-fixed N will result in huge environmental and economic benefits. To put this situation in some perspective, it has been estimated that ca. 288 Tg of fuel (at a cost of $30 billion U.S. annually) would be required to replace the N fixed by legumes with anhydrous ammonia produced by the Haber-Bosch process. For the U.S. alone, either decreasing or eliminating the use of synthetic N-fertilizers could save an estimated $1.0-4.5 billion annually (Tauer, 1989). This reduction in the amount of fuel consumed will also reduce undesirable impacts of the increased use of industrially-derived fix N. 3. SYMBIOTIC INTERACTION OF LEGUMES WITH RHIZOBIA In order to properly discuss how environmental stress factors influence symbiotic nitrogen fixation, it is important to understand how the micro- and macro-symbionts interact at the cellular and molecular levels. In some instances, environmental perturbation independently influences the nodulation and nitrogen-fixation processes. Although, in this chapter, I only discuss this topic in broad terms, several

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recent review articles cover this area in much greater detail (Hirsch et al., 2001; Roy et al., 2002; Vitousek et al., 2002). 3.1. The Nitrogen-fixation Process Environmental symbiotic N2 fixation requires the coordinate interaction of two major classes of genes present in rhizobia, the nif genes and fix genes. Only the nif genes, which encode the molybdenum-based enzyme system, are found in rhizobia and they have structural and functional-relatedness to the N2-fixation genes found in Klebsiella pneumoniae. The structural nif genes from taxonomically diverse microbes are nearly identical and function in a similar manner to encode nitrogenase (Ruvkin and Ausubel, 1980). A majority of the nif genes are plasmid borne in most rhizobia, but are located on the chromosome in the bradyrhizobia. Nitrogen fixation in symbionts and free-living microbes is catalyzed by nitrogenase, an enzyme complex encoded by the nifDK and nifH genes. Nitrogenase itself consists of a molybdenum-iron protein (MoFe), sometimes called component 1, and an ironcontaining protein (Fe), component 2. The MoFe protein subunits are encoded by nifK and nifD and an FeMo-cofactor (called FeMoco) is required for activation of the MoFe protein. This cofactor is assembled through the activity of the nifB,V,N,H and E genes. The Fe-protein subunit is encoded by the nifH gene. The organization and complexity of nif genes in microorganisms varies tremendously (Downie 1998). For example, in the free-living K. pneumoniae, at least 20 nif genes are organized in about 8 operons (Dean and Jacobson 1992). In most systems, however, the regulation of all nif genes is controlled by NifA (a positive activator of transcription) and NifL (the negative regulator). Environmentally, nif-gene expression is regulated by both O2 and fixed-nitrogen levels (Merrick and Edwards, 1995). For example, elevated soil ammonia (NH3 or NH4+) concentrations allow NifL to act as a negative controller of gene expression by preventing NifA acting as an activator. In addition, elevated O2 concentrations inhibit FixL from activating the transcriptional activator FixJ, which in turn prevents increases in NifA. Because NifA is the transcriptional activator of all other nif genes, elevated O2 results in a net decrease in the synthesis of nitrogenase and a decrease in, or abolition of, symbiotic N2 fixation (Monson et al., 1995). In addition to the nif genes, many other microbial genes are involved in symbiotic nitrogen fixation, these are collectively referred to as fix genes. Moreover, several other genes in the microsymbiont, including those for exopolysaccharide (Leigh and Walker, 1994; Glazebrook and Walker, 1989), hydrogen uptake (Baginsky et al., 2002), glutamine synthase (Carlson et al., 1987 ), dicarboxylate transport (Finan et al., 1983; Jiang et al., 1989), nodulation efficiency (Sanjuan and Olivares, 1989), β-1,2-glucans (Breedveld and Miller, 1994), and lipopolysaccharides (Carlson et al., 1987), either directly or indirectly influence symbiotic N2 fixation. Excellent in-depth reviews of the regulation of nitrogen fixation in free-living and symbiotic bacteria can be found in Merrick and Edwards (1995), Dean and Jacobson (1992), and Patriarca et al. (2002).

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3.2. The Nodulation Process The infection and nodulation process involves an intimate interaction of micro- and macro-symbiont, and is mediated by a bidirectional molecular communication between both symbiotic partners. The rhizobia induce two types of nodules on legumes, either determinant or indeterminant nodules (Franssen et al., 1992). The indeterminant nodules are most commonly formed on temperate legumes (pea, clover, and alfalfa) inoculated with the fast-growing nodule bacteria, whereas determinate nodules are normally induced by bradyrhizobia on tropical legumes, such as soybean and common bean. Rhizobia infect host plants, and induce root- or stem-nodules, using three fundamentally different mechanisms: (i) via root hairs (Kijne, 1992); (ii) entry through wounds, cracks, or lesions (Boogerd and van Rossum, 1997); or (iii) via cavities located around root primorida of adventitious roots (Boivin et al. 1997). In the root-hair mode of infection, rhizobia attach, often perpendicular, to susceptible root hairs within minutes of inoculation. Subsequent penetration of root-hair cell walls by rhizobia leads to root-hair curling, usually within 6-18 hours of inoculation. Rhizobia are enclosed within a plant-derived infection thread within the root-hair cell and move down the root hair towards the root cortex. Cell division within the root cortex, ahead of the approaching infection thread, eventually leads to the production of nodule primordia (Kijne, 1992). The infection thread spreads among cells of the nodule primordium and rhizobia are released into the host cortex by endocytosis. Within the host cytoplasm, the rhizobia are surrounded by a hostderived peribacteroid membrane, compartmentalizing the rhizobia into a symbiosome. Over time, the nodules expand and are usually visible 6-18 days after inoculation. Although nodulation initially is often heaviest in the crown of the root, secondary nodules frequently appear on lateral roots as the early crown nodules begin to senesce. The number and size of nodules on each legume host is controlled by the genotype of both the host and rhizobial partner, by the efficiency of the symbiotic interaction, by the presence of existing nodules, and by environmental factors, such as soil-nitrogen level and soil-moisture status (Caetano-Annoles, 1997; Sagan and Gresshoff, 1996; Singleton and Stockinger, 1983). 4. NODULATION AND NITROGEN-FIXATION GENETICS IN THE RHIZOBIA AND BRADYRHIZOBIA The majority of genetic studies on rhizobia and bradyrhiziobia have concentrated on the genetics and molecular biology of nodulation and N2 fixation. Over the last 17 years, advances in molecular biology and genetics have helped elucidated a large number of genes having symbiotic functions. In the fast growing species, symbiosis-related genes tend to be clustered on one or several relatively large symbiotic plasmids (Broughton et al., 1984; Hombrecher et al., 1981; Kondorosi et al., 1989), whereas in the bradyrhizobia, these genes are chromosomally located. The review articles listed below should be consulted for more detailed information on the genetics of nodulation and nitrogen fixation (Bladergroen and Spaink, 1998; Boivin et al., 1997; Debelle et al., 2001; Gualtieri and Bisseling, 2000; Pueppke,

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1996; Schultze and Kondorosi, 1998; Spaink, 1995; Spaink, 2000; van der Drift et al., 1998). Many of the bacterial genes involved in the formation of nodules on legumes have been identified and a complete description of the function of many of these genes can be found in several recent articles (Bladergroen and Spaink, 1998; Debelle et al., 2001; Niner and Hirsch, 1998; Pueppke, 1996). Taken together, more than 70 nodulation genes have been identified in rhizobia, although only a subset of these may be found in any single strain. Interestingly, despite this relatively large number, only a relatively few genes are required for nodulation of legumes (Göttfert, 1993; Long et al., 1985; Long, 1989; van Rhijn and Vanderleyden, 1995). Nodulation genes can be divided three broad groups based on their relationship to host specificity; they are common nodulation genes, hostspecific nodulation genes (hsn), and genotype specific nodulation (gsn) genes (Bachem et al., 1986; Bassam et al., 1986; Broughton et al., 1984; Davis et al., 1988; Djordjevic et al., 1985; Heron et al., 1989; Horvath et al., 1986; Lewin et al., 1987; Lie, 1978; Lewis-Henderson et al.,1991; Meinhardt et al., 1993, Nieuwkoop et al., 1987; Sadowsky et al., 1991; Sadowsky et al., 1995; Wijffelman et al., 1985). 4.1. Environmental Influences on Nodulation Genes As might be expected from the number of genes involved, the induction and repression of bacterial nodulation genes is under tight regulatory control and is a major factor influencing host specificity (Spaink et al., 1987) and response to environmental variables. The nodD gene can be viewed as a global regulatory gene which, together with plant flavonoid-signal molecules, activates transcription of other inducible nodulation genes (Banfalvi et al., 1988; Boundy-Mills et al., 1994; Djordjevic et al., 1987; Fellay et al., 1995; Göttfert et al., 1988; Innes et al., 1985; Kosslak et al., 1987; Long 1989; Long, 2001; Martinez and Palacios, 1990; Mulligan and Long, 1985; Olson et al., 1985; Peters et al., 1986; Price et al., 1992; Sadowsky et al., 1988; van Brussel et al., 1990; Zaat et al., 1987). The flavonoid nod-gene inducers are specific for a particular legume-Rhizobium interaction (Schlaman et al., 1998) and their production is influenced by environmental variables, like plant fertility, pH (Hubac et al., 1994), and Nod factors (Schmidt et al., 1994). Repressor proteins also play a role in nod-gene regulation (Kondorosi et al., 1988; Kondorosi et al., 1989; Kondorosi et al., 1991; Stacey et al., 2002). The nod genes of the microsymbiont are involved in the production of extracellular nodulation factors, called lipochitinoligosaccharides (LCOs) (Carlson et al., 1993; 1994; Downie, 1998; Lerouge et al., 1990; Pueppke, 1996). LCOs stimulate the plant to produce more nod-gene inducers, to deform root hairs on their respective host plant, and to initiate cell division in the root cortex (Banfalvi et al., 1989; Faucher et al., 1989; Lerouge et al., 1990; Price et al., 1992; Relic et al., 1993; Schultze et al., 1992; Spaink et al., 1991; van Brussel et al., 1990). Purified Nod factors have been shown to induce nodules on the specific host plant (Downie, 1998; Mergaert et al., 1993; Relic et al., 1993, Schultze et al., 1992; Truchet et al., 1991). The functions of nod genes and the basic structure of Nod factors for B.

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japonicum and several species of the genus Rhizobium have been reviewed (Carlson et al., 1993; Debelle et al., 2001; D’Haeze and Holster, 2002; Downie, 1998; Sanjuan et al., 1992). Recently, Endre and coworkers (2002) reported that a receptor kinase, NORK, in Medicago sativa is essential for Nod-factor perception in alfalfa and that the NORK system initiates a signal cascade, which leads to nodulation. The production of Nod factors by rhizobia is influenced by pH, temperature, and both phosphorus and nitrogen concentration (McKay and Djordjevic, 1993). Production and excretion of nod metabolites by Rhizobium leguminosarum bv. trifolii are disrupted by the same environmental factors that reduce nodulation in the field. Although much has been learned about legume nodulation over the last 7 years using genetic and molecular methods, the genomic revolution will no doubt substantially increase our understanding of this process at a very rapid rate. To date, the complete genomic sequence of the symbiotic plasmid from Rhizobium sp. strain NGR-234 (see http://genome.imb-jena.de/other/cfreiber/ pNGR234a2.html), S. meliloti (see http://cmgm.stanford.edu/~mbarnett/genome.htm and http://sequence.toulouse.inra.fr/meliloti.html), and the genomes of M. loti (see http://www.kazusa.or.jp/en/database.html) and B. japonicum (see http://www.genome.clemson.edu /~twood/projects/brady.html and http://www.kazusa.or.jp/en/database.html, and http://www.kazusa.or.jp/rhizobase/Bradyrhizobium/index.html) have been determined. For further information, please also see volume 3 of this series entitled Genomes and Genomics of Nitrogen-fixing Organisms. 5. RHIZOBIA IN THE SOIL ENVIRONMENT Rhizobia can exist in two fundamentally different modes. They can live in soils either as free-living saprophytic heterotrophs or as legume-host-specific nitrogenfixing symbionts. This dual mode of existence gives rhizobia several distinct advantages with respect to survival and persistence over most other soil bacteria. The bulk soil that surrounds legumes contains relatively large numbers of rhizobia, often approaching 106 cells g-1 in soils of the American Midwest (Ellis et al., 1984) and sometimes up to 108 cells g-1 (Bottomley, 1992). The growth of rhizobia in the rhizosphere may be stimulated by plant root exudates (Van Egeraat, 1975), although more research is needed in this area. Interestingly, Phillips et al. (1999) reported that rhizobia can also stimulate growth and respiration of leguminous plants. Generally speaking, rhizobia in soils are associated with aggregates (Mendes and Bottomley, 1998; Postma et al., 1990), which gives them some degree of protection from perturbations by environmental and biotic factors. However, the nodule environment affords the rhizobia a unique niche in which to multiply while being protected. Nodules can contain more than 1010 rhizobia g-1 (McDermott et al., 1987). Nodule senescence at the end of the growing season leads to the release of a large number of rhizobia into soils. Numerous studies have shown that a legume host is not needed for persistence (saprophytic competence) of rhizobia in soils (Bottomley, 1992; Brunel et al., 1988; Chatel et al., 1968; Kucey and Hynes, 1989). Although nodule bacteria and bacteroids, after release into the environment,

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are often susceptible to osmotic and other soil stress factors (Sutton, 1983), many of the released rhizobia survive and persist in the soil as free-living, heterotrophic saprophytes for long periods until they again come into contact with susceptible legume host (Diatloff, 1977; Brunel et al., 1988; Lindstrom et al., 1990). 6. STRESS FACTORS IN THE SOIL ENVIRONMENT THAT INFLUENCE N2 FIXATION Virtually any environmental factor that negatively influences either the growth of rhizobia or the host plant itself has a dramatic impact on symbiotic N2 fixation. These factors can independently negatively influence the nodulation process itself, and thereby indirectly affect nitrogen fixation, or directly influence plant growth and vigor during post-nodulation events and so affect the efficient functioning of the nitrogenase enzyme complex. To facilitate discussion, I have separated these fundamentally different sets of factors below. However, the reader should be aware that, in some instances, some factors simultaneously affect both rhizobia and the host plant. 6.1. Soil Water Content and Stress Soil water influences the growth of soil microorganisms through processes of diffusion, mass flow, and nutrient concentration (Paul and Clark, 1988). Soil water is related to soil pore space, and soils containing larger pores and pore spaces retain less water. Thus, soil aggregates having smaller internal pore spaces are more favorable environments for the growth of rhizobia and most soil microbes (Papendick and Campbell, 1981; Turco and Sadowsky, 1995). Soil-water content also directly influences the growth of rhizosphere microorganisms, like rhizobia, by decreasing water activity below critical tolerance limits and indirectly by altering plant growth, root architecture, and exudations. Poor nodulation of legumes in arid soils is likely due to decreases in population levels of rhizobia during the dry season. However, the influence of soil-water activity on plant growth and vigor, and hence nodulation, should not be ignored. Water activity (Aw) and water potential (ȥ) values are parameters often used for describing water relations with respect to microorganisms (Harris, 1981). Rhizobia vary widely in their tolerance to water stress, and there is little apparent correlation to taxonomic criteria and phylogenetic relationships. Although bradyrhizobia have been suggested to be more resistant to water stress than the rhizobia (Bushby and Marshall, 1977), the reverse has also been reported (Mahler and Wollum, 1981). In solute-controlled water stress conditions, such as those controlled by salts, bacteria are able to grow and survive when Aw values are in the range of 0.76 - 0.99. This range corresponds to water potential (ȥ) values of -15 to -350 bars. In rhizobia (and all bacteria), both cell membranes and walls play a pivotal role in tolerance to water-potential stress. Under conditions of elevated salt or other osmolytes, water stress is dominated by the movement of water in response to the ȥ gradient (Harris, 1981). The total ȥ of a microbial cell is due to the sum of the ȥ from components of intermediary metabolism, and the intracellular accumulation of stress and

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compatible solutes. Many microbes, including rhizobia, accumulate compatible solutes, such as amino acids, salts, and betaines, as a means to equilibrate internal and external osmotic concentrations (Csonka, 1991; Csonka and Epstein, 1996). Osmoregulation is a complex problem for rhizobia because they must be able to adapt to unfavorable and changing environmental conditions, as well as to osmoticstress conditions associated with the infection process itself and life in the nodule. The detrimental effects of salt stress on inoculum viability, nodulation and nitrogen fixation have been reported for many Rhizobium spp. strains (Israel, 1988). Thus, elevated osmotic conditions can limit symbiosis by affecting survival and proliferation of rhizobia in the soil, inhibiting the infection process, or by directly affecting root-nodule function. Rhizobia have evolved a variety of mechanisms for adapting to osmotic stress, mostly by the intracellular accumulation of inorganic and/or organic solutes. For example, R. meliloti overcomes osmotic stress-induced growth inhibition by accumulating compatible solutes, such as K+, glutamate, proline, glycine betaine, proline betaine, trehalose, and the dipeptide, N-acetylglutaminylglutamine amide (Bernard et al., 1986; Botsford, 1984; Botsford, 1990; LeRudulier and Bouillard, 1983; Smith et al., 1988; Boscari et al., 2002). Some compatible solutes can be used as either nitrogen or carbon sources for growth, suggesting that their catabolism may be regulated to prevent degradation during osmotic stress (Smith and Smith, 1989). 6.2. Desiccation Tolerance Some species of Rhizobium are also susceptible to non solute-mediated desiccation, referred to here as matric-mediated drought conditions. Species of Rhizobium differ in their susceptibility to the detrimental effects of desiccation in natural soils. Slowgrowing rhizobia are generally thought to survive desiccation in a sandy soil better than fast-growing types (Bushby and Marshall, 1977; Bushby and Marshall, 1977a). Mahler and Wollum (1980; 1981) reported that moisture level was the dominant factor influencing both short- and long-term survival of B. japonicum strains inoculated into a loamy sand. Both soil type and temperature are important factors influencing survival of rhizobia in desiccated soils (Boumahdi et al., 2001; Mahler and Wollum, 1980; Trotman and Weaver, 1995). There is no information available concerning the genetics and physiology of desiccation resistance in rhizobia, however, Boumahdi et al. (2001) reported that a reduction in medium Aw led to a decrease in unsaturation of cellular fatty acids in rhizobia (Boumahdi, et al., 2001). On addition to soils, rapid moisture loss is responsible for rhizobial death on the seed surface (Vincent et al., 1962). In addition, and generally speaking, rhizobia are more resistant to soil-water deficit (drought) than the plant itself. Nevertheless, rhizobial strains that are superior under drought conditions have been reported (Athar and Johnson, 1996; Athar and Johnson, 1997; Hunt et al, 1981). The impact of drought conditions on N2 fixation is also due to direct influences on the plant partner (see reviews by Serraj et al., 1999 and Hungria and Vargas, 2000). Soil-water deficit influences several aspects of the host legume, including but not limited to, nodule establishment, carbon and nitrogen metabolism,

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nitrogenase functioning, and photosynthetically-derived energy supply. Drought stress should be thought of as influencing the host in a global manner, rather than a collection of individual processes. Results from studies over the last 30 or so years have shown that the N2-fixation process itself is more sensitive to drought conditions than gas exchange from leaves (Durrand et al., 1987; Sinclair et al., 1986), the accumulation of plant dry matter and carbon assimilation (Sinclair et al., 1987; Wery et al., 1994), photosynthesis, and nitrate assimilation (Purcell and King, 1996; Wery et al., 1988). However, as discussed above, soil moisture also indirectly influences total plant N2 fixation by decreasing nodule mass and number (Sangakkara et al., 1996; Sinclair et al., 1988). The decline in nodule number under drought conditions is most likely due to impacts on the infection process itself. Root-hair infections and infection-thread formation are negatively influenced by drought conditions (Sprent, 1971; Graham, 1992). 6.3. Nutrient Stress As might be expected, soil nutrient status has a tremendous influence on the symbiosis, as well as on the independent growth and survival of both partners. It should be noted, however, that in some cases, nutrient stresses are indirectly caused by changes in soil matric potential or acidity, which limit nutrient bioavailability, rather than to the lack of the presence of nutrients per se. When considering nutrient limitations to symbiotic nitrogen fixation, one must clearly separate factors affecting growth of the host from those influencing the microbe or the symbiotic interaction. For example, acid and water stress causes alterations in root growth, which can indirectly affect both nodulation and nitrogen fixation. This effect is thought to be mediated by abscisic acid (Zhang et al., 2001). Stress conditions apparently increase requirements for essential elements, such as Ca2+, P, and N, in both plants and microsymbionts (Beck and Munns, 1984; O’Hara et al., 1989; Zahran, 1999). Ca2+ might, in some instances, offset the deleterious influence of low pH on root growth and ion uptake (Torimitsu et al., 1985) and increase nod-gene induction and expression (Richardson et al., 1988). Calcium deficiency, with or without the confounding influence of low pH, also affects attachment of rhizobia to root hairs (Caetano-Anolles et al., 1989; Smit et al., 1992), and nodulation and nodule development (Alva et al., 1990). Lastly, a calcium-spiking phenomenon is initiated in root-hair cells of legumes by nodulation factors and rhizobia (Wais et al., 2002), suggesting that Ca2+ plays a pivotal role in symbiotic interactions at the molecular level. Phosphorous supply and availability remains a severe limitation to nitrogen fixation and symbiotic interactions. About 33% of the ariable land in the world is limited by P availability (Graham and Vance, 2000; Pereira and Bliss, 1989; Sanchez and Euhara, 1983). This situation is especially true in soils impacted by low pH. There are marked differences in rhizobial and plant requirements for P (Beck and Munns, 1985; Pereira and Bliss, 1989) with the slow-growers being more tolerant to low P than the fast-growing rhizobia (Beck and Munns, 1985). Nitrogenfixing plants have an increased requirement for P over those receiving direct nitrogen fertilization, owing perhaps to nodule development and signal transduction

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(Graham and Vance, 2000), and to P-lipids in the large number of bacterioids. Moreover, in white lupin, P deficiency leads to enhanced P acquisition by the formation of proteoid roots (Johnson et al., 1996), changes in carbon metabolism, enhanced secretion of citrate and malate from roots, and release of a novel acid phosphatase into the rhizosphere proteoid roots (Gilbert et al., 1999). Nodules themselves are sinks for P (Hart, 1989) and nodulation and N2 fixation are influenced strongly by P availability (Leung and Bottomley, 1987; Singleton et al., 1985; Saxena and Rewari, 1991). The rhizobia respond to P stress in a manner analogous to the host plant; that is, low P induces expression of genes involved in P acquisition (phosphatases, phosphate transporters) and acidification of the root zone (Al-Niemi et al., 1997; Smart et al., 1984; Torriani-Gorini et al., 1987). In addition to macro nutrients, the growth and persistence of rhizobia in soils is also influenced by several other nutritional factors (reviewed by Brockwell et al., 1995 and Bottomley, 1992). The rhizobia are metabolically diverse and have been shown to use a variety of both plant- and soil-derived compounds for growth. Interestingly, some of the same compounds that support growth have also been shown to be chemotactic and induce nod genes (Parke and Ornston, 1986; Sadowsky and Graham, 1998). In addition, supplementation of soil and inoculants with gutamate, glycerol, and organic matter has been shown to enhance the survival and numbers of rhizobia in soils and increase both early nodulation and N2 fixation (Rynne et al., 1994). This result indicates that, although rhizobia can surely persist in soils, their efficacy can be enhanced by carbon addition, which suggests that they are C limited in the natural state.

6.4. Soil pH Stress The influence of soil pH on the nodulation process has been extensively examined, in part due to the World’s large number of acid soils (see reviews by Graham, 1992 and Hungria and Vargas, 2000). Worldwide, more than 1.5 Gha of acid soils limit agriculture production (Edwards et al., 1991; Graham and Vance, 2000) and as much as 25% of the earth's croplands are impacted by problems associated with soil acidity (Munns, 1986). Brockwell et al. (1991) reported a nearly 10-3 decrease in the number of S. meliloti in soils with a pH < 6 compared to those with a pH > 7.0. As found for soil moisture, there is a range of effects of soil pH on rhizobia, but relatively few grow and survive well below pH values of 4.5-5.0 (Graham et al., 1994; Vargas and Graham, 1988). Competitive interactions have been shown to be influenced by soil pH. Generally speaking, the bradyrhizobia are more acid tolerant than the rhizobia (Brockwell et al., 1991; Date and Halliday, 1979; Keyser and Munns, 1979; Sadowsky and Graham, 1998), although some strains of R. tropici are very acid tolerant (Graham et al., 1992). The influence of soil pH on the behavior of rhizobia in soils can be dramatic. Generally, S. fredii is more competitive than B. japonicum in soybean nodulation in neutral soils in Spain, but B. japonicum USDA 110 outcompeted S. fredii in soil at pH 4.9 (Triplett and Sadowsky, 1992). Furthermore, despite the fact that R. etli is more competitive than R. tropici in nodule formation with beans (Chaverra and Graham, 1992; Martinez Romero and Rosenblueth,

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1990), acidification of soils led to the replacement of R. etli by introduced, acidtolerant R. tropici (Anyango et al., 1995; Hungria et al., 1997). However, the relationship between soil acidity, competitiveness, and the ability to survive in acid soils is not always straightforward and due to resistance to acidity. For example, Richardson and Simpson (1989) reported that many rhizobia from acid soils are sensitive to acidity. They suggested that soil microniches protect these rhizobia from extremes of soil pH. So, merely isolating a rhizobial strain from root nodules of plants grown in acid conditions does not guarantee that the isolated strain will be acid resistant (Graham, 1992). Furthermore, metals, such as Al, Cu, and Mn that become more soluble at lower pH, may also secondarily contribute to the inhibition of the growth and persistence of rhizobia in acid soils (Cooper et al., 1983; Coventry and Evans, 1989; Hungria and Vargas, 2000; Lal, 1993; Reeve et al., 2002). Moreover, soil Ca and P levels are also influenced by soil pH (Bell et al., 1989; Munns, 1970) and may secondarily influence the growth and survival of rhizobia. Nevertheless, results from several studies have indicated that tolerance to acid conditions in rhizobia is often correlated to the strains ability to maintain an internal pH approaching neutrality (pH 7.2-7.5) (Graham et al., 1994; Kashket, 1985; O’Hara et al., 1989). This ability has been suggested to be due to proton exclusion (Graham, 1991), enhanced cytoplasmic-buffering capacity (Krulwich et al., 1985), the presence of acid-shock responses (Bhagwat and Apte, 1989), the presence of glutathione (Riccillo et al., 2000), the maintenance of elevated cellular potassium and glutamate concentrations (Aarons and Graham, 1991; Graham et al., 1992), membrane permeability (Chen et al., 1993), and calcium metabolism (Howieson et al., 1992). Although the microsymbiont appears more pH sensitive than the host partner (Hungria and Vargas, 2000), acidity also influences both the growth of the legume plant and the infection process (Munns, 1986). This effect is, in part, most likely due to both a disruption of signal exchange between macro- and micro-symbionts (Hungria and Stacey, 1997) and repression of nodulation genes and excretion of Nod factor in the rhizobia (Richardson et al., 1988). Interestingly, nodulated legumes appear more sensitive to metal toxicity by Mn and Al than to their N-fed control counterparts (Hungria and Vargas, 2000). Recently, using molecular techniques and proteomics, Glenn and colleagues have shown that rhizobial genes, such as actA, actP, exoR, lpiA, actR, actS, and phrR, are essential for growth at low pH (Dilworth et al., 2001; Glenn et al., 1999; Reeve et al., 2002). Vinuesa and coworkers (2003), using a Tn5-mutagenesis approach, isolated and characterized pH-responsive genes, lpiA and atvA, from Rhizobium tropici CIAT899. Complementation analyses indicated that atvA, an ortholog of the A. tumefaciens acvB gene, is required for acid tolerance. 6.5. Soil Temperature Temperature has a marked influence on survival and persistence of rhizobial strains in soils. For example, cowpea rhizobia from the hot dry Sahel-savannah of West Africa grow at 37oC, and more than 90% of the strains isolated from this region grew well to 40oC (Eaglesham et al., 1991). The influence of temperature on

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rhizobia appears to be both strain and soil dependent. For example, Bradyrhizobium sp. (lupins) was less susceptible than R. leguminosarum bv. trifolii to high soil temperatures, but addition of montmorillonite and illite remediated this problem in sandy soils (Marshall, 1964). Soil temperature also greatly influences competition for nodulation (Kluson et al., 1986; Triplett and Sadowsky, 1996). This effect may, in part, be due to a temperature-induced delay in nodulation or the restriction of nodules to the sub-surface region (Munns et al., 1977). Both temperature extremes need to be examined when considering the influence of soil temperature on the growth and survival of rhizobia, and the latter two parameters need to be separated. For example, whereas rhizobia isolated from temperate regions often survive at 4oC, little growth occurs at this temperature (Trinick, 1982). Nevertheless, there have been reports of growth of rhizobia from the Canadian high arctic at 5oC (Prévost et al., 1987) and 10oC (Caudry-Reznick et al., 1986). However, the symbiosis itself is sensitive to low temperatures; cooler root-zone temperatures limit nodulation and nitrogen fixation in the soybean-B. japonicum symbiosis (Lynch and Smith, 1994; Zhang et al., 1995). There are a few generalities that can be made. Although high soil temperatures may lead to death of many rhizobia isolated from temperate climates, strains from tropical region generally survive better at high soil temperatures. Nevertheless, Somasegaran et al. (1984) reported that incubation of inoculant strains at 37oC led to a gradual decline in population levels over an 8-week period. Some temperaturetolerant Bradyrhizobium sp. strains that nodulate cowpea in Nigeria have also been noted (Hartel and Alexander, 1984). In addition, temperature-tolerant strains of rhizobia can be either artificially (Karanja and Wood, 1988; Hartel and Alexander, 1984) or naturally selected for (Zahran et al., 1994). However, excessive temperature shock has been shown to cure plasmids in fast-growing strains and some strains, which were isolated from high-temperature environments, have a Fix– phenotype (Hungria and Franco, 1993; Moawad and Beck, 1991). Effective hightemperature (40oC) tolerant rhizobia that are capable of nodulating and fixing nitrogen with Phaseolus vulgaris (Hungria et al., 1993; Michiels et al., 1994), Acacia (Zerhari et al., 2000), and Prosopis (Kulkarni and Nautiyal, 1999) have also been reported. Although there have been several attempts to adapt rhizobia to higher temperatures for inoculation of legumes in tropical regions, in most cases, incubation of strains at elevated temperatures results in the loss of either infectivity or effectivity (Segovia et al., 1991; Wilkins, 1967). Relatively high-root temperature has also been shown to influence infection, N2fixation ability, and legume growth (Arayankoon et al., 1990; Hungria and Franco, 1993; Kishinevsky et al., 1992; Michiels et al., 1994; Munevar and Wollum, 1982) and it has a strong influence on specific strain and cultivar interactions (Arayankoon et al., 1990; Munevar and Wollum, 1982). It appears that every legume/Rhizobium combination has an optimum temperature relationship, which is around 30oC for clover and pea, between 35-40oC for soybean, peanut and cowpea, and between 2530oC for common bean (Michiels et al., 1994; Piha and Munns, 1987). Exposure of both symbiotic partners to temperature extremes much above or below these critical temperatures impairs infection, nodulation, nodule development, and general nodule functioning (Gibson, 1971; Roughley, 1970) as well as both plant growth and

SOIL STRESS FACTORS AND SYMBIOTIC NITROGEN FIXATION 101 productivity. High soil tempratures also restrict nodulation to sub-surface regions where cooler temperatures prevail (Graham, 1991). The physiological basis feor the temperature sensitivity of the symbiosis is most likely complex because many cellular functions in both host and microbe are affected by elevated and low temperature. Nevertheless, elevated temperature directly influences the production or release of nod-gene inducers from soybean and bean (Hungria and Stacey, 1997), it alters nodule functioning due to leghemoglobin synthesis, nitrogenase activity, and hydrogen evolution, and, in addition, hastens nodule senescence (Hungri and Vargas 2000). Although heat-shock proteins have been found in rhizobia (Aarons and Graham, 1991; Labidi et al., 2000; Michiels et al., 1994; Munchbach et al., 1999) and heat stress alters the mobility of LPS (Zahran et al., 1994), their direct role in either heat tolerance or sensitivity has not been demonstrated. 7. CONCLUDING REMARKS Despite many decades of progress and the acquisition of a large amount of useful information, the physiological and molecular bases for the tolerance of legumemicrobe symbiotic systems to environmental stress remains largely unknown and empirical in nature. Although understanding these processes was originally thought to be straightforward and tractable, we have learned that we now have more questions than answers. This situation is perhaps due to the fact that abiotic stresses independently and differentially influence the host legume, the rhizobia, and the symbiotic couple. So where do we go from here? Clearly, more work needs to be done on the underlying molecular bases for tolerance to stress factors in both legume and microbe. Recent advances in the genomics and proetomics of macro- and microsymbionts will accelerate progress in this area by providing a wealth of information on how both host and microbe respond to environmental perturbations. For example, proteome analysis has been used to investigate oxidative stress in the Rhizobium etili-Phaseolus vulgaris symbiosis (del Carmen Vargas et al., 2003), to define bacterial genes involved in growth at low pH (Dilworth et al., 2001; Glenn et al., 1999; Reeve et al., 2002), and to examine cultivar-specific interactions between Rhizobium leguminosarum bv. trifolii and subterranean clover (Morris and Djordjevic, 2001). Similarly, Saalbach and coworkers (2002) and Wienkoop and Saalbach (2003) have used proteome analysis to investigate the proteins in the pea and lotus peribacteroid membrane, respectively, and Mathesius et al. (2001) have established a root proteome reference map of Medicago truncatula that can be used with expressed sequence tag databases (Fedorova et al., 2002; Lamblin et al., 2003) to investigate molecular mechanisms of root symbioses in legumes. This type of global organismal information at the genomic and proteomic levels, however, now needs to be coupled to traditional plant breeding and microbial selection efforts in order to rapidly define and utilize microbial and host genetic loci that are involved in tolerance to a large number of environmental stresses.

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Chapter 7

NODULATED LEGUME TREES J. I. SPRENT 32 Birkhill Avenue, Wormit, Fife DD6 8PW Scotland, UK

1. INTRODUCTION The Leguminosae is the third largest family of flowering plants. One of its most widely recognised features is the ability of many species to form a symbiotic relationship with soil bacteria, leading to the formation of nitrogen-fixing root or (rarely) stem nodules. This chapter considers only those legume genera that contain tree species. As many legume trees lack the ability to nodulate and many others have not been checked for this ability, the emphasis is on the known nodulating genera. Because the information on these genera is not readily available in a concise form, a major part of the chapter will be on generic descriptions. Following this section, both nodule characteristics and nodulating bacteria will be discussed. Because mycorrhizal formation is an integral part of the nutrient-uptake systems and is important in nature and for management, these will also be briefly considered. A consideration of the problems of measuring nitrogen fixation by trees will precede a discussion of the role of nitrogen-fixing legume trees in natural ecosystems and how they may be exploited in a sustainable way. 2. LEGUMINOSAE Although the way in which legumes should be classified is generally agreed, the terminology is not. Many people still prefer to use Leguminosae as the family name, but for reasons of historical precedence, Fabaceae is now widely accepted. Within either of these terms, three subfamilies are usually recognised, although some workers endow these with family status. Thus, we may have Caesalpinioideae (Caesalpiniaceae), Mimosoideae (Mimosaceae) and Papilionoideae (Papilionaceae). In this chapter, I shall use Leguminosae and three subfamilies. The tribes and genera within the subfamilies are also generally agreed on, although some workers still retain 113 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 113-141. © 2005 Springer. Printed in the Netherlands.

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the tribe, Swartzieae, within the Caesalpinioideae, rather than the more common position in the Papilionoideae. The spelling of some names has changed over the years. Here, the spelling agreed to and used by the International Legume Data Base and Information Service (ILDIS, web site at http://www.ildis.org/LegumeWeb/) will be used. Earlier spellings can be found in Sprent (2001). Table 1 summarises the main features of the subfamilies and indicates the number of genera with confirmed reports of nodulation. Throughout this chapter, only those genera and species, where nodulation has been confirmed, will be discussed. Many reports of swellings on roots, which appear superficially to be nodules, have not been confirmed, for example, on the tropical tree genera, Eperua and Mora. Examples of such structures are illustrated in Sprent (2001). Criteria used in establishing nodulation include appearance, location on roots, internal coloration and structure, nitrogenase activity, isolation of rhizobia and establishment of Koch’s postulates, and immunochemical location of key enzymes, particularly nitrogenase. Not all of these criteria have been used in all cases, but a minimum of three is considered by the author to be essential. An additional complication is that many legumes, particularly tropical trees are difficult to identify. Where specimens have been lodged in herbaria, it is possible to check identification at a later stage and this procedure is strongly recommended. It is essential, when giving either generic or specific names, to quote the authority for these names (as here) because so many have been subjected to name changes and a single species may still be known by several names, under more than one genus. Table 1. Major Features of Legume Subfamilies.

Number of genera - approximate total Confirmed as nodulating Distribution Habit Flower/infloresence

Nodulation

Caesalpinioideae 157

Mimosoideae 78

Papilionoideae 479

8 Mainly moist tropics Mainly trees

42 297 Tropics/subtropics, Tropics to arctic, often in dry areas dry to flooded Mainly trees and Trees, shrubs, shrubs and herbs Zygomorphic, Regular-frequently Classically peawith marked but very variable in globose or standard and spicate heads with wings prominent stamens Rare, nodule Common, but with Very common, with some structure usually important exceptions primitive exceptions

Many woody legumes, particularly in the tropics, produce lenticels on stems. These have been recorded as nodules, e.g., on Cassia tora, now classified as Senna tora (Subba Rao and Yatazawa, 1984), and have shown nitrogenase activity. Recent evidence suggests that nitrogen-fixing bacteria may be found in airspaces in such lenticels, but that these are not symbiotic (E. Wang, personal communication) and

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they will not be included in this chapter. There are no confirmed reports of stem nodules on trees, although many trees produce adventitious roots on even large trunks and, in humid forests, these roots may be abundantly nodulated, e.g., Pterocarpus species in Cameroon as illustrated in Sprent (2001). With very few exceptions, which will be discussed later, nodulation appears to be a generic character. Indeed, genera that have recently been sub-divided on classical taxonomic grounds have proved to be coincident with either the presence or absence of nodules, e.g., Chamaecrista (nodulating) from both Cassia and Senna (nonnodulating), and Sophora (nodulating) from Stypholobium (non-nodulating). Further details and references on these genera can be found in Sprent (2000; 2001) and Lavin et al. (2001). In the following taxonomic sections, details of nodulating species are from Sprent (2001). Numbers in brackets following many entries are for species that are either potentially or actually threatened and are taken from Oldfield et al. (1998). However, it should be noted that the latter is not an exhaustive text as many species remain to be evaluated. Other references are given for particular genera. Many genera are composed of both shrubs and small trees, whereas others have a broader range of habits. The distinction between a shrub and a small tree depends not only on the species, but also on the habitat, so they have not been treated separately here when both forms occur within a genus. For most of the genera, information on uses and tree size has been taken from Allen and Allen (1981). It is very surprising that very few tree legumes (rather than the more shrubby species used in agroforestry), with the major exception of Acacia species, have been either planted or managed with a view to maximising their nitrogen-fixing potential (Sprent and Parsons, 2000). For this reason, I have tried to highlight some genera that should be more widely studied. In some cases, attention has been drawn to them by the excellent books produced by the US National Academy of Sciences, for example, Anon (1979). With current concerns over deforestation and the need for low-input agriculture and forestry, perhaps more consideration will be given to nodulated legume trees. In order to paint as complete a picture of them as possible, the first sections will cover all genera known to have nodulated trees as members, arranged alphabetically within subfamilies. In this section, only references not included in Sprent (2001) are cited. 2.1. Caesalpinioideae Nodulation is rare in this subfamily, as has been known for many years (Allen and Allen, 1981). However, there are still many genera that have not been examined for nodules. Nodules are always of indeterminate growth. Except for some shrubs and all herbaceous species of Chamaecrista (Naisbitt et al., 1992) that have been examined, the nodules have bacteroids retained within infection-thread-like structures, now usually termed fixation threads, throughout the period of nitrogen fixation (Faria et al., 1987). The genera are confined to certain tribes within the subfamily, details of which can be found in Sprent (2001).

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2.1.1. Campsiandra Benth. Now considered to have 22 species of both shrub and tree, one of which is known to be nodulated, this genus is mainly found in inundated regions, such as the Amazon and Orinoco basins. It is relatively unusual in having functional nodules under water (Sprent, 1999). Some species have durable timber and some are used medicinally. 2.1.2. Chamaecrista Moench. This genus currently contains 287 species, making it the largest nodulating genus in the Caesalpinioideae. It is unusual in having a wide range of forms, from trees to annual herbs, and in being the only genus in the subfamily with nodulating species (all herbaceous) in temperate areas. Of the 47 species known to nodulate, a few are trees. These may fix N2 in their natural habitat (Sprent et al., 1996; Perreijn, 2002), the only caesalpinioid species for which such evidence is available. 2.1.3. Dimorphandra Schott. This genus has 26 species of tree of various heights. It is found in tropical S America, where it is widespread. Nine of these species are reported as nodulated and several are potentially endangered. This genus is sometimes included in the subfamily, Mimosoideae, e.g., in Lorenzi (1992). Although there is little available information on its uses, some species have beautifully colored wood (Ribeiro et al., 1999). 2.1.4. Erythrophleum Afzel. ex G.Don. This genus has eight species of tree in Africa and Asia plus one in Australia. Three of the African species are known to nodulate and there is indirect evidence that the Australian species may do so. Apart from some herbaceous pantropical species of Chamaecrista, this is the only caesalpinioid genus outside the Americas with confirmed ability to nodulate. The typical caesalpinioid structure of infected cells has recently been shown for three African species (Sprent, 2001; A. Kiam et al., unpublished data). Species are used for timber and pharmaceutical purposes and at least one (E. fordii from Asia) is in such demand for timber that it is listed as threatened. 2.1.5. Melanoxylon Schott. The genus has two species in tropical Brazil, one of which, M. brauna, has been reported to nodulate. This species has very fine, long-lasting timber and, although not listed as threatened, it is now becoming very scarce. 2.1.6. Moldenhowera Schrad. The seven species of this genus are found in tropical Brazil and Venezuela and one has been reported to nodulate. Its timber is used for construction purposes and it is also widely grown as an ornamental (Lorenzi, 1992).

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2.1.7. Sclerolobium Vogel. This genus of forty species of tree is found in northern tropical South America. Ten species have reports of good nodulation. Some are extensively exploited for timber and five species are threatened. 2.1.8. Tachigali Aubl. From tropical central and South America, there are reports of good nodulation for three of the 24 species, with one species threatened. 2.2. Mimosoideae Although nodulation is more widespread in this subfamily, it is by no means universal (Table 2). A group of the basal genera appears not to nodulate. Arguably, they never had this character, whereas other less-basal genera may have lost the ability to nodulate (Sprent, 2001). The taxonomy of this subfamily has been the subject of major revisions in recent years. These changes have been incorporated as far as possible by Sprent (2001) and the same classification will be used here. As with the previous section, this section will take the genera in turn, alphabetically. There are many tree genera for which there are no reports of nodulation and these are not included here, but their details are available elsewhere (Sprent, 2001). Table 2. Some Mimosoid Tree Genera that Appear to Lack the Ability to Nodulate.

Calpocalyx Harms Cylicodiscus Harms Dinizia Ducke Newtonia Baill.

Parkia R.Br Tetrapleura Benth Zapoteca H.M.Hern

2.2.1. Abarema Pittier This genus of 44 species, mainly trees, is from the New World tropics. It is largely made up of species formerly included in Pithecellobium Mart, but also includes some species from other genera. Ten species have been reported as nodulated. Nodules are indeterminate, but not as freely branched as in Acacia. The genus is not widely used for timber, but some species have medicinal properties and others are used for tannin. Twenty species are threatened. 2.2.2. Acacia Mill. This is by far the largest genus of nodulating legume trees. It is also one of the three legume genera known to have non-nodulating species. These sixteen (so far) closely related non-nodulating species, in the sub-genus Aculeiferum, have probably lost the nodulation character because some of the relevant genetic material has been retained (Sprent, 2001). So far, no Australian species has been confirmed not to nodulate. This fact is, perhaps, not surprisingly because the Australian species are generally quite distinct from the African and American species that do not nodulate.

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It is likely that, in the next few years, the genus will be divided into a number of individual genera, rather than the three subgenera now commonly used (Table 3), but this process is proving to be rather difficult. However, there are several differences in symbiotic properties that relate to geographical origin as well as to subfamily. In particular, many Australian species have both arbuscular and ecto- mycorrhizae, an important property in terms of their ability to be exploited for land reclamation and also become invasive weeds (see later). Nodules on Acacia are indeterminate, often considerably branched. They vary greatly in effectiveness, a fact that is probably related to the wide range of rhizobia that nodulate many species (see later). Table 3. Features of the Subgenera of Acacia. (Phyllodineae is sometimes known as Heterophyllum).

Subgenus

Number of species

Main distribution

Characters

________________________________________________________ Acacia

120-130

Pan tropical

Aculeiferum

180-190

Pan tropical, Bipinnnate leaves; with mainly Africa prickles or unarmed and New World

Phyllodineae

>900

Largely Australian

Bipinnate leaves; stipular spines; some have swollen spines associated with ants

Bipinnate leaves in seedlings; usually phyllodes in adults; no prickles; some have stipular spines

Almost all species are shrubs or trees. Many occur as understory plants in forests, particularly eucalypt forests in Australia; others can develop either into large trees in open moist areas, e.g., A. xanthophloea Benth. in Africa or into smaller trees in extremely dry habitats, e.g., some species in the Negev desert in the Middle East. These species are important in many ways. They are used as very high quality timber (e.g., A. melanoxylon R.Br. in temperate Australia), as fuel wood (many African species), as forage and browse for wild and domestic animals (many African species), for honey (almost all species from dry areas), as food additives (gum arabic from A. senegal (L.) Wild.), for tannins (from bark of various species, particularly A. mearnsii De Wild.), and in land reclamation (dual mycorrhizal Australian species, such as A. auriculiformis Benth.). Thirty-six species, from various parts of the world, but in particular dry areas of Africa, are listed as potentially threatened. 2.2.3. A lbizia Durazz. This genus is currently undergoing taxonomic changes, but now consists of about 120 species, many of which are trees. They are most numerous in the Old World tropics, especially Africa. Forty-four species are reported to nodulate and their nodules are

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abundant and often profusely branched. Some species produce valuable timber, others medicinal products, but possibly the best known use is as shade for crops, such as cocoa and coffee. Sixteen species are potentially threatened. 2.2.4. Anadenanthera Speg. This genus consists of two species of large tree, native to tropical South America, of which one has been reported to nodulate, with much branched nodules (Gross et al., 2002). It produces excellent timber and has a number of other uses. It is not to be confused with the related genus, Adenanthera L., which cannot nodulate. 2.2.5. Archidendron F. Muell. A genus that is sometimes included with Pithecellobium. It has 94 species of shrub or tree and is native to Indo-Malaysia and parts of Queensland, Australia. Four species have been reported as nodulated with nodules that are branched and indeterminate. Some species are used for timber and three species are threatened. 2.2.6. Archidendropsis I. Nielsen This genus is a segregant from Albizia and contains 14 species of tree from the SouthWest Pacific area. One species has been reported to have indeterminate nodules and three are listed as threatened, largely as a result of habitat clearance. 2.2.7. Balizia Barneby and Grimes Another segregant of Pithecellobium, this genus has three species occurring in wet tropical New World areas, one of which has been reported to nodulate. 2.2.8. Calliandra Benth. Currently, this genus has 132 species of shrub and small tree from tropical or subtropical America. Fourteen species have been reported as nodulating and the shrubby/small tree, C. calothyrsus Meisn., is now being widely used in agroforestry (see later). Madagascan and South Asian species have now been transferred to Viguieranthus Villiers, but species combinations for all of these are not yet published, so it is not possible to give their nodulation status. The genus is not in the latest version of ILDIS (6.05, July 15 2002), but see Du Puy (2001). 2.2.9. Cedrelinga Ducke A monotypic genus of nodulated tree from Amazonia and one of the largest to grow there, reaching a height of 50m. 2.2.10. Chloroleucon (Benth.) Britton and Rose The genus consists of ten species of shrub and tree, which were formerly included in the Pithecellobium, and are native to tropical America. Four species are reported to

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nodulate with indeterminate branched nodules. Some species are xeromorphic and five are potentially threatened. 2.2.11. Cojoba Britton and Rose Another segregant of Pithecellobium, this genus one has twelve species of shrub or tree in tropical America, one of which has been reported to have indeterminate nodules. 2.2.12. Dichrostachys (A.DC.) Wight and Arn Currently, this genus contains 14 species of shrub or small tree, which are native to the Old World tropics. It is, however, thought to be polyphyletic, so the number of species may well be reduced in future, if some are transferred to other genera. The species, D. cinerea, nodulates and has a number of subspecies. It can be very invasive when introduced into new areas, e.g., in some of the forest reserves in Cuba. On the other hand, two species from Yemen, Somalia and Ethiopia are at risk. 2.2.13. Elephantorrhiza Benth. Fourteen species of shrub or small tree, which are native to tropical and South Africa, constitute this genus. Four species are known to nodulate and have indeterminate branched nodules. 2.2.14. Entada Adans. Twenty-five to 30 species of shrub, small tree or liana, which are found in tropical America, Africa and Australia, make up this genus. Seven species are recorded as nodulated. Some have pods that are over 1m long and are said to have been used as clubs by policemen in the West Indies. 2.2.15. Enterolobium Mart. This genus consists of ca. ten species of tree in tropical America, eight of which have been reported to nodulate. Many of the trees are large (40m high) with spreading canopies and broad buttressed trunks. They are used for many purposes, however, no species is currently listed as threatened. It is surprising that this fast-growing tree has not been more developed for commercial purposes, especially as their potential has been recognized (Anon, 1979). 2.2.16. Falcataria (Nielsen) Barneby and Grimes Three species of tree, one of which nodulates, make up this genus from the Pacific area and Queensland. All species have been included in other genera in the past. 2.2.17. Havardia Small Five species of tree from warm America, previously classified under Pithecellobium, constitute this genus, with one species having indeterminate, branched nodules.

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2.2.18. Hesperalbizia Barneby and Grimes A monotypic nodulated tree from Central America, formerly included in Albizia. 2.2.19. Hydrochorea Barneby and Grimes Four species of shrub or tree, formerly included in Pithecellobium, with one able to nodulate. 2.2.20. Inga Mill. This large and important genus of shrub or tree from tropical and subtropical America has recently been the subject of much taxonomic study. Currently, about 350 species are recognised. Fifty-six species have been reported to nodulate; nodules in some species start out more or less spherical, but later branch. A monograph has been published of its many uses (Pennington and Fernandes, 1998), which include that of a shade tree for coffee, where its ability to fix nitrogen is considered important (van Kessel, 1983). 2.2.21. Faidherbia A. Chev. This monotypic nodulating genus, also known as Acacia albida Del., is native to Africa and has many of the same uses as Acacia, plus the additional useful property of shedding its leaves in the wet season and producing them in the dry season. 2.2.22. Leucaena Benth. About 22 species, which occur in areas ranging from Texas south to Peru, make up this genus. Nodulation has been reported for five species. One species, L. leucocephala (Lam) de Wit, has been used extensively in agroforestry because of its many useful properties, which include a degree of drought and salinity tolerance, plus the ability to nodulate freely and fix significant amounts of nitrogen. Unfortunately, these same characters, coupled with its ability to produce large numbers of seed, have led to it becoming an invasive weed in many areas where it has been introduced. Calliandra calothysus (see above) is now usually preferred for agroforestry in many areas, especially in Africa (Kanmegne and Degrande, 2002). 2.2.23. Lysiloma Benth. Currently, this genus is considered to have about nine species, with many others having been reassigned to other genera, such as Acacia. Recognised species are shrubs and trees native to Central America and the West Indies. Five have been reported to nodulate and the nodules are indeterminate. 2.2.24. Macrosamanea Britton and Rose ex Britton and Killip A genus of 11 species, previously classified under Pithecellobium or Inga, which are shrubs or slender trees found in seasonally flooded soils in tropical South America. They are, therefore, of interest for the possible flood tolerance of nitrogen fixation. Five have been reported to nodulate with branched indeterminate nodules.

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2.2.25. Mimosa L. This large genus (ca. 480 species) has habits ranging from herbs to large trees. Fifty species can nodulate. The nodules are indeterminate and often branched. M. caesalpinifolia Benth. is xerophytic and widely used for timber, fuel, and charcoal production and is one of three tree species listed as threatened. M. scabrella Benth. lacks root hairs and rhizobial infection occurs between epidermal cells, but other species are infected classically via root hairs (E. K. James, personal communication). 2.2.26. Mimozyganthus Burkhart A monotypic nodulated xerophytic shrub or tree from Argentina. 2.2.27. Parapiptadenia Brenan Six species of shrub or tree from tropical eastern Brazil make up this genus. Two species have been reported as nodulated. 2.2.28. Paraserianthes I.C.Nielsen Currently, this genus has two tree species from Malaysia and tropical Australia, which were previously assigned to other genera. One species has indeterminate nodules. 2.2.29. Pentaclethra Benth. The genus has only two or three species and is one of those known to have variable nodulation. There is good evidence that the African species, P. macrophylla Benth., does not nodulate and equally good evidence that the south America P. macroloba does. The genus is thought to be fairly basal within the Mimosoideae and close to some of the non-nodulating genera listed in Table 2, but some taxonomists believe that these two species are definitely from the same genus. They produce good timber, have protein-rich edible (after cooking) seeds, and medicinal uses. 2.2.30. Piptadenia Benth. About 21 species of tree, shrub or liana, found from Mexico south through Peru to Argentina, constitute this genus. Six species have been reported to nodulate and one of the Peruvian species is listed as threatened. 2.2.31. Piptadeniastrum Brenan A monotypic large tree from West Africa, which has indeterminate branched nodules, produces excellent, though odoriferous, timber that has some termite resistance. 2.2.32. Pithecellobium Mart. Now a much smaller genus than previously constituted, it has about 18 species of shrub or tree from tropical America. Six species have been reported to nodulate and seven are threatened.

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2.2.33. Plathymenia Benth The two species of shrub or tree that make up this genus are found in tropical South America; both are nodulated. Although small, the trees produce fine wood, which is much exploited and, for this reason, one species is listed as threatened. 2.2.34. Prosopis L. Forty-four species of shrub and tree from various warm parts of the world, particularly Central America, constitute this genus. Sixteen species are reported to nodulate. Some species are among the most drought- and salt-tolerant woody plants known. They are exploited for agroforestry and land reclamation. They are used widely for wood, honey, medicines, tannin, forage, hedges, and many other purposes. 2.2.35. Pseudosamanea Harms This is a monotypic genus of one nodulated tree from Central and South America. In Sprent (2001), P. guachapele is given as nodulated. This species is now accepted in Albizia with the one remaining species of Pseudosmanea, P. cubana (Britton and Rose) Barneby and Grimes, having no record on nodulation, but listed as threatened. 2.2.36. Pseudopiptadenia Rauschert This genus has eleven species of shrub or tree, which range from northern South America to southern Brazil. The two nodulating American species of Newtonia Baill., an otherwise non-nodulating genus from Africa, have been transferred to a single species, P. contorta (DC.) G.P.Lewis and M.P. Lima, and a second species is also known to nodulate. 2.2.37. Samanea Merr. This genus is thought by some to be part of Albizia. It has three species of tree from northern South America. Two of these species are reported to nodulate, including S. saman (Jacq.) Merr., the so called ‘rain tree’, which closes its leaves when it rains. This tree can grow to great heights, over 60m, and its canopy may cover 0.2ha. It is found on many different types of soil and, although widely grown, is not exploited in a major way for commercial purposes. 2.2.38. Schleinitzia Warb. Of its four species of shrub or tree from the Pacific region, formerly included in other genera, one is reported to nodulate. 2.2.39. Stryphnodendron Mart. About 30 species of tree from Central and tropical South America, of which four are known to nodulate, constitute this genus.

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2.2.40. W allaceodendron Koord This monotypic nodulated tree from the Philippines and Sulawesi, produces excellent dark wood. 2.2.41. X erocladia Harv. This monotypic nodulated shrub or small tree occurs in Namibia and South Africa. 2.2.42. Zygia P. Browne This genus contains many species that have been transferred from Pithecellobium and other genera. The current number of species is 57, mainly trees and shrubs from tropical America. Ten are known to nodulate and two are listed as threatened. 2.3. Papilionoideae This is generally considered as the most advanced subfamily. It consists mainly of small shrubs and annual or perennial herbs, however, there is a significant number of trees, some of which cannot nodulate (Table 4). Like those in the Mimosoideae, most of the non-nodulating genera are considered to be in the basal part of the Papilionoideae. This section covers those genera with tree species known to nodulate. As with other subfamilies, there are numerous genera for which there are no reports. Table 4. Some Papilionoid Tree Genera that Appear to lack the Ability to Nodulate.

Aldina Endl. Alexa Moq. Amburana Schwacke and Taub. Amphimas Pierre ex Harms Angylocalyx Taub. Castanospermum A. Cunn. ex Hook. Cladastris Raf. Cordyla Lour. Dipteryx Schreb. Exostyles Schott Harleyodendron R.S.Cowan

Lecointea Ducke Luetzelburgia Harms Milbraediodendron Harms Myroxylon L.f Pterodon Vogl Styphnolobium Schott ex Engl Taralea Aubl Vatairea Aubl. Vataireopsis Ducke Xanthocersis Baill. Zollernia Wied.-Neuw. and Nees

2.3.1. A cosmium Schott This genus has been the subject of taxonomic research, together with the related genus, Sweetia Sprengl. At present, Acosmium is considered to have 17 species of shrub or tree from tropical America. Reports on nodulation are very varied, but the evidence for A. nitens (Vogel) Yakovlev is strong. Nodules are indeterminate and may be found under water (Barrios and Herrera, 1994).

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2.3.2. A ndira Juss. It includes thirty species of tree, occurring in tropical America and West Africa. Reports on nodulation have been mixed, but there are ten positives that have been confirmed. One of the problems here is that the nodules vary greatly in size and are usually black and woody on the outside, even when small. Further, their internal structure is of the primitive type, typical of the Caesalpinioideae, with bacteroids retained within fixation threads. These factors make nodules difficult to recognise and also make isolation of rhizobia tricky. Trees may be large; many are attractive and a good source, inter alia, of honey, timber and drugs. This genus has more potential than has yet been realised. One species is threatened. 2.3.3. Baphia Afzel. ex Lodd. Forty-six species of shrub, tree or liana, found in tropical Africa, South Africa and Madagascar, constitute this genus. Three species are reported to nodulate. Nodules are indeterminate but, unusually, may have lenticels when young. Some species produce useful wood and other products, such as dyes, and sixteen are threatened. 2.3.4. Bergeronia M. Micheli This genus consists of a monotypic nodulated shrub/small tree from northern South America. 2.3.5. Bobgunnia J.H. Kirkbr. and Wiersema This genus of two species was recently segregated from Swartzia; one is nodulated. 2.3.6. Bolusanthus Harms This monotypic nodulated tree from tropical South Africa has insect-resistant wood. 2.3.7. Bowdichia Kunth Two nodulated species of tree from tropical South America make up this genus. The trees may grow up to 50m in height and produce course, heavy wood with good resistance to decay. 2.3.8. Brongniartia Kunth. Currently, this genus contains about 56 species of shrub and tree, which are found on drier soils from Texas south through Central America to the Andes. Surprisingly, only one has been reported to nodulate so far. Their natural habitats suggest that they could be useful additions to the known suite of woody plants for dry, alkaline areas. 2.3.9. Brya P. Browne This genus has twelve species of shrub or small tree from the West-Indian region. Two species are known to nodulate with nodules of the aeschynomenoid type (see below).

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2.3.10. Calia Berland This segregant of Sophora consists of six species of shrub or tree from Mexico and the southern USA. One species is reported to nodulate. 2.3.11. Callerya Endl. The genus has recently been enlarged to include species from other genera, including Millettia. It currently consists of about 19 species of shrub, tree and liana from East and South-East Asia and Australia. Two species have been reported to nodulate. 2.3.12. Calpurnia E. Mey This genus has seven species of shrub or small tree that are found in southern India and in the eastern Cape region of South Africa. Three species have been reported as nodulating. 2.3.13. Centrolobium Mart. ex Benth. Of these six species of tree from South America, three have been reported to nodulate with nodules of the aeschynomenoid type. The wood is high quality and is used especially for furniture. One species is threatened. 2.3.14. Chadsia Bojer This genus of about 17 species of shrub or small tree is confined to Madagascar. One species has been reported to have indeterminate branched nodules. 2.3.15. Chamaecytisus Link A genus of 30 species of shrub or small tree, which is native to Europe and the Canary Islands, was formerly included in Cytisus Desf. Six species have been reported to nodulate. There are unusual features both in the infection by rhizobia and in nodule development of the shrubby species C. proliferus Link (Vega-Hernàndez et al., 2001). 2.3.16. Clathrotropis (Benth.) Harms It consists of six species of tree from tropical South America, of which two are reported to nodulate. Although the trees produce hard wood, it has not proven useful commercially. 2.3.17. Coursetia DC. There are thirty-eight species of shrub and small tree, all are found in the warmer parts of America. Three species have been reported to nodulate, including the tall tree species, C. ferruginea (Kunth) Lavin, which is useful for wood. Two species are on the threatened list, including one that may produce compounds effective against the AIDS virus.

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2.3.18. Cullen Medik. This genus has undergone various taxonomic changes recently. It currently contains about 36 species ranging from annual herbs to small trees, which are widely distributed in the Old World tropics. Four species are known to nodulate. 2.3.19. Cyclolobium Benth. Now considered as a monotypic genus, it is nodulated with bacteroids retained within fixation threads. 2.3.20. Dahlstedtia Malme This genus consists of two species of shrub or small tree that are found in Argentina and Brazil. One species has been reported to nodulate with bacteroids retained within fixation threads. 2.3.21. Dalbergia L.f. This is a large genus of tree, shrub or liana from tropical regions. All species examined to date nodulate with aeschynomenoid nodules (see below) that can be formed on quite old roots as long as they retain the potential to form laterals. Foresters, however, have shown very little interest in the nitrogen-fixing activity of this genus (Sprent and Parsons, 2000). Many of the tree species produce very fine timber, which is widely exploited. Seventy-two species are on the threatened list. 2.3.22. Dalbergiella Baker f. This genus consists of three species of shrub or small tree that are found in tropical Africa, one of which has been reported to nodulate. 2.3.23. Dalea L. A few of the 160 species of this genus are trees and at least some of these are known to have indeterminate nodules. They are found from southern Canada down to Argentina. Plants are browsed by wild mammals and have some local uses, for example, in basket making. 2.3.24. Dendrolobium (L.) Benth. These are twelve species of shrub or small tree found in Indo-Malaysia and northern Australia. Three have been reported as nodulating. This is one of the few papilionoid tree genera from the tribe Desmodieae (now being included in the Phaseoleae). Their nodules are of determinate growth. 2.3.25. Derris Lour. Recently the subject of many taxonomic changes, the genus currently consists of 5060 species, most of which are either shrubs or lianas, but a few are trees. Of the

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species reported to nodulate, only three remain in the genus, one of which is a tree. Best known for the insecticidal properties of the roots of some species, tree species have been used on a local scale for many purposes, such as culinary, turnery, shade and green manure. 2.3.26. Diphysa Jacq. This genus has fifteen species of shrub or small tree from tropical America, mostly in the uplands, with one species reported as nodulating. 2.3.27. Diplotropis Benth. Four of the twelve species of tree of this genus from tropical South America are reported to nodulate. The larger tree species produce excellent wood. 2.3.28. Erythrina L. This is a large genus of about 120 species of shrub or tree with pantropical distribution. It is the only genus, which has large trees, that is found in the tribe, Phaseoleae, which contains important grain legumes, such as soybean and cowpea. Like them, its nodules may be determinate although, in Erythrina, they sometimes become lobed. The tree species usually have brilliant flowers, but the seeds are poisonous and their toxins have been used as fish poisons. Wood of the genus is of limited use, but it has been exhaustively studied for its alkaloids and other pharmaceuticals. Some research has been performed on its nitrogen-fixing potential (see references in Sprent and Parsons, 2000), but there is scope for much more. Twelve species are on the threatened list. 2.3.29. Etaballia Benth This monotypic nodulated tree from tropical South America produces high-quality wood. 2.3.30. Eysenhardtia Kunth. About 10 species of shrub or small tree from Central America constitute this genus with one species reported as nodulating. Some species produce good timber and several are browsed by animals, such as cattle and horses. 2.3.31. Geoffroea Jacq. It consists of two species of shrub or tree from tropical South America, the West Indies, and Panama, one of which is reported to nodulate. 2.3.32. Gliricidia Kunth This is one of the few tree genera, which have been intensively studied for features that include nitrogen fixation. It has four species of tree from tropical America,

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which are fast growing, produce a variety of marketable products, and are widely used in agroforestry in Africa and elsewhere. 2.3.33. Harpalyce Moçino Sessé This genus has about 24 species of shrub or tree, which grow in hot dry and often rocky areas of Mexico, Cuba and Brazil. This genus presents another opportunity for species that could be useful for management of dry areas. Two species have recently been reported to have indeterminate nodules. 2.3.34. Hebestigma Urb. This monotypic nodulated tree grows in Cuba and its wood is used locally. 2.3.35. Hesperolaburnum Maire This monotypic nodulated tree is found in the mountains of Morocco. 2.3.36. Hymenolobium Benth. This genus has about 12 species of tree from tropical South America, of which six have been confirmed to nodulate. Nodule bacteroids are retained within fixation threads, as in the Caesalpinioideae. Many species produce strong timber. This is a genus with high potential. 2.3.37. Kotschya Endl. This genus consists of 30 species of herb or small tree from tropical Africa; five have been reported to nodulate with aeschynomenoid nodules. Some tree species occur up to 2000m altitude, a comparatively rare feature among nodulated legume trees. 2.3.38. Laburnum Fabr. Both species of nodulated tree from southern Europe have toxic seeds and are widely grown as ornamentals. The fine textured wood is little exploited commercially. 2.3.39. Leptoderris Dunn From about 20 species of shrub or small tree, which originate from tropical West Africa, only one has been reported as having indeterminate nodules. 2.3.40. Lonchocarpus Kunth. This large genus, which currently has about 130 species of shrub or tree, originates mainly from tropical and subtropical America, but with one species in Africa. Twenty-one species have been reported as nodulated and in one species, at least, nodulation is unusual in that infection occurs between epidermal cells. The wood is not considered to be a commercially viable product, but the genus is well known for the range of insecticidal products it produces. Thirteen species are threatened.

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2.3.41. Maackia Rupr. With just six species of tree from East Asia and East Siberia, one has been reported to have indeterminate nodules. This genus produces heavy dark wood that is used in construction. One species is threatened. 2.3.42. Machaerium Pers. This fairly large genus, containing about 120 species of tree and liana, originates mostly from tropical America, but with one species in West Africa. Twenty-four species have been reported to nodulate. Tree species are relatively few in number and not widely exploited for commercial use. Five species are threatened. 2.3.43. Millettia Wight and Arn Currently, with ca. 150 species of tree, shrub and liana, the genus is probably polyphyletic and may be divided in the future. It is found in the Old World tropics and 12 species appear to nodulate. Like its relative, Lonchocarpus, Millettia species produce insecticides and other toxic agents. Some produce good timber and most have attractive flowers. It can be found in dense rainforests, where it may play an important role. Twenty-nine species are on the threatened list. 2.3.44. Mundulea (DC.) Benth. Twelve species of shrub or tree from Madagascar and other parts of the Old World tropics make up this genus. One species has been reported to nodulate. Members of this genus produce various toxins, including insecticides. 2.3.45. Olneya A. Gray This monotypic nodulated genus of shrub or small tree from south-western North America has hard heavy wood that is used locally. 2.3.46. Ormocarpum P. Beauv. Twenty species of shrub and small tree from tropical and subtropical areas of the Old World, three of which have been reported as nodulated, constitute this genus. It is used as browse, for shade, and other purposes. Three species are threatened. 2.3.47. Ormosia G. Jacks About 145 species of shrub or tree from tropical America, Asia and Australia, of which twelve species are reported to nodulate, make up this genus. Nodules may be very large, woody, and profusely branched. 2.3.48. Ortholobium C.H. Stirt. Formed from part of Psoralea, the genus has 53 species of herb, shrub, or tree, which are found in East and South Africa and in Soouth America. Six nodulate.

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2.3.49. Paramachaerium Ducke It consists of five species of tree from tropical South America, one of which has been reported to nodulate and another is on the threatened list. 2.3.50. Pericopsis Thwaites Previously classified as Afrormosia Harms, all three species, two of which are from tropical Africa and one from Sri Lanka, are nodulated. Nodules are indeterminate. The trees produce fine wood that has been widely used as a substitute for teak. For this reason, two of the species are listed as threatened. This genus appears to be an excellent target for properly managed plantation use, taking into account its ability to fix nitrogen, which until now has been neglected. 2.3.51. Philenoptera Fenzl. ex A. Rich. These twelve species of tree, shrub or liana are from tropical Africa and Madagascar and were previously mainly included in Lonchcarpus. Two are known to nodulate. 2.3.52. Piscidia L. Of the about seven species of shrub and, occasionally tree, found in Florida, Central America, and south to northern Peru, only two species are known to nodulate. 2.3.53. Platycyamus Benth. Of two tree species from tropical South America, one is reported to nodulate. 2.3.54. Platymiscium Vogel Consisting of eighteen species of tree from tropical Central and South America, seven so far have been reported as nodulated with nodules of the aeschynomenoid type. Some of the trees exceed 30m in height with a girth of 10m. They have showy flowers and produce high quality timber. Two are threatened. 2.3.55. Platypodium Vogel It consists of only one or two species of tree from Central and South America, one of which is reported to nodulate. 2.3.56. Poecilanthe Benth. This genus has about ten species of shrub or tree (two threatened) from tropical South America. Five species nodulate with bacteroids retained within fixation threads. 2.3.57. Psoralea L. Now restricted to 50 species of herb, shrub or tree from South Africa (see also Ortholobium above), this genus has 27 species that are known to nodulate.

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2.3.58. Pterocarpus Jacq. This pan-tropical genus of tree has both nodulated and non-nodulated species. The latter are confined to Brazil, even though Brazilian soils have rhizobia that can nodulate African species. Sixteen species are reported to nodulate, with nodules of the aeschynomenoid type. Trees may be very large with buttress roots and even large trees may be profusely nodulated in the Cameroonian rain forests (see below). A characteristic feature of the genus is that, when cut, the xylem exudes a bright red sap. Wood of the African and Asian species is highly prized. Six species are threatened. 2.3.59. Robinia L. This genus has 4 species of shrub or tree from North America. R. pseudoacacia L. is nodulated and has been widely introduced into other areas, such as central Europe. It fixes nitrogen well, but also suckers freely and may become invasive. It produces useful wood and is a good source of honey, but some parts produce substances toxic to animals, such as cattle and sheep. 2.3.60. Sophora L. This genus now has about 50 species of herb, shrub or tree, after other species, including all the non-nodulating ones, were transferred to Styphnolobium. Nineteen species have been reported to nodulate. Nodules are indeterminate. Many woody species are grown for the flowers but they also have many other uses, although some have leaves that are toxic to cattle, sheep and goats. Eight species are threatened. 2.3.61. Swartzia Schreb. This genus has about 150 species of tree, shrub or liana, most of which are found in tropical South America, but with some in tropical Africa and Madagascar. Twentyfive species can nodulate. Nodules are indeterminate and may grow quite large. The trees are very ornamental and some species produce compounds used to stupefy fish. At present, they do not appear to have much commercial potential, although they may be important in their ecosystems. Seven species are threatened. 2.3.62. Tipuana (Benth.) Benth. This monotypic genus of nodulated tree from subtropical South America has nodules that are aeschynomenoid. It is a fast-growing species that has been introduced to many countries where it is used for various purposes ranging from ornamental to carpentry, tanning and dying. 2.3.63. V irgilia Poir. Two species of tree, both reported to nodulate, constitute this genus from the Cape region of South Africa.

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2.3.64. X eroderris Roberty This monotypic genus of nodulated tree from tropical Africa is used as stock feed, for making canoes, and in tanning. 3. RHIZOBIA THAT NODULATE LEGUME TREES Other chapters in this book series cover rhizobial taxonomy. Here, I shall only cover the most important general considerations for trees and then give some information specific to particular trees. Most of the nodulated legume trees are currently found in tropical/subtropical regions. The widest range of studies on their rhizobia has been by Moreira and co-workers, using mainly Brazilian species. However, I believe that their finding that there is no close correlation between host and rhizobial genotypes is of general application (Moreira et al., 1998). This research group is the only one so far to consider members of all three subfamilies, but a number of others has looked at members of the Mimosoideae and Papilionoideae. Bala and Giller (2001) isolated rhizobia from soils in seven counties on three continents and looked at their ability to nodulate the woody legumes, Calliandra calothyrus, Leucaena leucocephala, and Sesbania sesban. As with earlier workers, most notably Barnet and Catt (1991) with Australian Acacia species, they found that soil properties were just as important as the host in determining which strains were present and potentially nodulating. Further, in contrast to much of the earlier work cited in Allen and Allen (1981), these and other workers have found that many rhizobia, which nodulate tropical trees, are fast- as well as slow-growing. There are numerous reports that host range and nodule effectiveness vary greatly with both host species and rhizobial strain. One of the first papers in this area was that of Turk and Keyser (1992), but their work has been supplemented by that of others from several continents, including Bala and Giller (2001), Burdon et al. (1999), and Odee et al. (2002). The overall picture is that many trees may be nodulated by a variety of rhizobial genera and species, but that some have a greater degree of host specificity than others. This range of specificity is found within host genera. Further, within a host species that is nodulated by many rhizobia, there is a wide range of effectiveness; from ineffective, where nodules are essentially parasitic, to highly effective, where nodulated plants may fix more nitrogen than can be assimilated by control plants on combined nitrogen (e.g., Burdon et al. (1999) for work with Australian acacias). Nodule mass on plants with ineffective strains may be far greater than on plants with effective strains. For examples, see McInroy (1997) and the illustration on p. xii of Sprent (2001). A further point to stress is that nodule structure is host determined. This was first shown for peanut and cowpea, both of which can be nodulated by the same strain of slow-growing Bradyrhizobium, where in the former case, the nodules are aeschynomenoid and the export product of nitrogen fixation is amino acids, but in the latter, the nodules are desmodioid and export ureides (Sen and Weaver, 1984). This observation has since been extended to trees, for example, caesalpinioid species with fixation threads and mimosoid species without these structures (Moreira et al., 1998). There are numerous examples of soils from one country having rhizobia that will nodulate exotic species. These include Pterocarpus (as mentioned above), where

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rhizobia in Brazilian soils nodulate African species, even though the Brazilian species cannot nodulate (Faria and Lima, 1998) and the Australian acacias, A. auriculiformis and A. holosericea, which nodulate with slow-growing rhizobia in Kenya whereas native species nodulate with fast-growing rhizobia (Odee et al., 1998). Most of the detailed work on rhizobia that nodulate trees has been carried out with species used in agroforestry and land reclamation/soil amelioration, although the large genus Acacia has been studied more widely in both Africa and Australia. Many species of Acacia may be nodulated by both fast- and slow-growing rhizobia, although one type tends to predominate in a particular soil. A. senegal L. Willd. is one of the more promiscuous species known, being nodulated by at least 6 species of rhizobia from the three genera, Mesorhizobium, Rhizobium, and Sinorhizobium (Nick, 1998). Within the genus, there may be differences, for example, the wide-host-range NGR234, which can nodulate members of all three subfamilies, only formed effective nodules on the largely Australian sub-genus, Phyllodineae, but it could form effective nodules on the related African species, Faidherbia albida (Pueppke and Broughton, 1999). Even within Australian species, Burdon et al. (1999) found wide variation in host/rhizobial specificity and effectiveness. These problems have led to the suggestion that multi-strain inoculants may sometimes be preferable to single strains (Sutherland et al., 2000). A rather similar situation obtains for Calliandra, although most of the available data are only for C. calothyrsus. This species is nodulated by a range of both fastand slow-growing rhizobia, but with the latter predominant and including species similar to those of the genera, Agrobacterium, Mesorhizobium, Rhizobium and Sinorhizobium (Bala and Giller, 2001). As with Acacia, effectiveness varies widely. It is not nodulated by NGR 234 (Pueppke and Broughton, 1999). One of the few temperate species to be studied in detail is Robinia pseudoacacia, which is also nodulated with both fast- and slow-growing rhizobia (Batzli et al., 1992; Röhm and Werner, 1992). 4. TYPES OF NODULE FORMED ON TREES Legumes have been known for many years to have a range of nodule morphological types and these were found by Corby (e.g., 1988) to have taxonomic value. Taking internal structure into consideration as well as morphology, the categories used by Corby were modified by Sprent and co-workers. The categories found in trees, which do not include the lupinoid form, are given in Table 5. The majority of legume nodules from all latitudes have indeterminate growth, but significant groups have determinate forms. One of the reasons that complicated the development of the classification of Corby was the discovery that all genera (but not all species, see above) in the Caesalpinioideae and some in the Papilionoideae do not release bacteroids into symbiosomes in the active nitrogen-fixing stage, but instead retain them in fixation threads. Many of the indeterminate nodules on trees are woody and often have a more extensive vascular system than their herbaceous counterparts (Sprent et al., 1989).

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Table 5. External Features of Nodule Types found in Tree Legumes. For further detail, see Sprent (2001).

Type Features Examples ___________________________________________________________________ Aeschynomenoid

Associated with lateral roots; determinate, oblate.

Dalbergia, Pterocarpus

Desmodioid

Determinate, with lenticels.

Dendrolobium, Erythrina

Indeterminate

Apical meristem, often branched, sometimes extensively.

Common in all subfamilies

5. MYCORRHIZAS AND OTHER NUTRIENT-ACQUISITION SYSTEMS The mycorrhizal status of legume trees is almost certainly important for the optimal functioning of nodules (see Barea et al., this volume). Nodulated legume trees may be arbuscular mycorrhizal (AM), ectomycorrhizal, or both. However, there are distinct variations with both taxonomic position and with geographical location. Within the Caesalpinioideae, all confirmed cases of ectomycorrhizal species lack the ability to nodulate. Arbuscular mycorrhizal species may or may not nodulate. Interactions between AM and nodulation have not been studied in this sub-family. Classical ectomycorrhizas, with a Hartig net, have not been reported in the Mimosoideae and are rare in the Papilionoideae. Some shrubby endemic Australian legumes are both ectomycorrhizal and nodulated. In Africa, the papilionoid species, Pericopsis angolensis (Baker) van Meeuwen, in some areas forms ectomycorrhizas and then does not nodulate; in other areas, it forms AM and may then nodulate (Högberg and Piearce, 1986), thus confirming an early observation by Bonnier (1960) that African legumes are rather versatile in their symbioses. Some Australian species of Acacia produce both AM and also a form of ectomycorrhiza, which is less highly structured than the classical type, particularly in having a much looser fungal sheath. Purists may not accept this structure as a true ectomycorrhizal structure but, because it is formed using standard ectomycorrhizal fungal species, many workers accept them as a form of ectomycorrhiza. Dual mycorrhizal acacias are confined to parts of the sub-genus Phyllodineae and all can nodulate. Some, e.g., A. mangium Willd., are important timber species that have been introduced into Africa, Brazil, and elsewhere. In these countries, they can associate with both AM and ectomycorrhizal fungi from the local soil, although native acacias are only AM. The symbioses of these acacias allow them to grow in very poor soils (see below), but the ectomycorrhizal partner may also confer other benefits, such as reducing the plant parasitic nematode populations (Founoune et al., 2002). There are numerous publications showing that AM and nodulation may be complementary for acquiring nutrients in legume trees. The overall picture is similar to that in crop and

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pasture legumes, where AM may help plants obtain P from poorly available sources (Sprent, 1999; Giller, 2001; and references therein). Cluster roots occur in a number of shrubby legumes of Australia and South Africa, some of the former also have both AM and classical ectomycorrhizas. An early suggestion that cluster roots form on acacias has recently been confirmed for A. mangium in the Pilbara region of Western Australia (P. Landsman and M. Adams, personal communication). Compared with the cluster roots found on either shrubby legumes or non-legume trees, such as species of Grevillea R. Br. ex J. Knight, those on acacias are very small and their significance remains to be established. They may emerge as being more common among acacias and to be important in accessing poorly available soil nutrients. 6. MEASUREMENT OF NITROGEN FIXATION BY TREES Accurate measurement of nitrogen fixation in large field-grown trees is virtually impossible. Thus, attempts to quantify nitrogen fixation in natural ecosystems and to understand its role in them are still at a very early stage (Vitousek et al., 2002). General methods of measuring nitrogen fixation are considered elsewhere in these volumes (see Dilworth, in Volume 1, Catalysts for Nitrogen Fixation); of these, the acetylene-reduction assay is useful for confirming that nodules are nodules and that they are active. Nitrogen-balance methods are useful when soil N is very low and the 15 N-dilution method has been shown to be useful for work on potted trees or young trees in plots and agroforestry systems. For example, Ståhl et al. (2002) used this method to compare the effects of nodulated trees as fallows in a maize crop-rotation system in Kenya. Sesbania sesban (L.) Merr. fixed 280-360 kg N ha-1 and Calliandra calothyrsus Meisn. 120-170 kg N ha-1 in 22 months, which are figures comparable to those for crop legumes. In natural ecosystems, the measurement of 15N at natural abundance is the only feasible method currently available. Although it has its problems, in some circumstances and when used with care, it can give very useful data (Boddey et al., 2000). Sprent et al. (1996) used it to show nitrogen fixation in a number of woody plants from the Cerrado region of Brazil. Perreijn (2002) made the first attempt to use the method in combination with a number of others to look at the magnitude of different reactions in the nitrogen cycle in a lowland tropical rainforest in Guyana. He concluded that there was wide variation in the amount of nitrogen fixed by various nodulated trees from areas with different soils and other conditions. In West Africa (Côte d’Ivoire), Galiana et al. (2002) used the method successfully to show variations in nitrogen fixation by Acacia mangium in soils of different nutrient status. 7. ROLE OF LEGUME TREES IN NATURAL AND MANAGED SYSTEMS 7.1. Tropical Rain Forests. There appears to be a longitudinal gradient in the occurrence of legume trees in tropical rain forests, with fewest in Asian forests. In addition, the proportion of those

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legume trees with the potential to nodulate increases when going from Asia via Africa to tropical America. In Africa, most legume trees lack the ability to nodulate but, in Amazonia, a significant number can (Sprent, 2001). However, recent unpublished work by the author and others in Cameroon, supported by the work of Perreijn (2002) in Guyana, suggests that nodulated legumes may play a bigger part than once thought. Contrary to perceived wisdom that mature trees recycle most of their nitrogen and, therefore, need few nodules, extensive networks of nodulated roots were observed on some very large trees and lianas in Cameroon and on lianas, in particular, in Guyana. Thus, we have the interesting possibility that a few trees may fix nitrogen and share it, possibly via mycorrhizas, with other trees in the ecosystem. If this is the case, then selective logging could be even more disastrous than now thought. Many tropical rainforests are close to major rivers. In South America, nodulated legumes trees from all sub-families are found bordering the Orinoco, often with their nodules being under water (Sprent, 1999). Shrubby legumes, such as Discolobium pulchellum Benth., in the flooded Pantanal of Brazil, may only nodulate on stems when submerged (Loureiro et al., 1994). The role of submerged nodules in tropical ecosystems is a subject needing further study. 7.2. Legume Trees in Dry and Saline Areas. The genera, Acacia and Prosopis L., have many species that can grow and nodulate in dry and/or saline soils. For example, in the Negev desert, almost the only trees to be found are species of Acacia and these may enhance soil-nutrient status but, at the same time, may increase soil salinity (Zuzana and Ward, 2002). Some aspects of management of such trees will be considered in the next section. 7.3. Management of Nodulated Legume Trees. In traditional forestry, nitrogen fixation by legumes has largely been ignored (Sprent and Parsons, 2000), even though many legume genera, e.g., Dalbergia spp., produce valuable timber (see above). In agroforestry, land reclamation, and other situations, nitrogen fixation and other attributes of legume trees are considered to be important for management and some examples are given below. For the humid tropics, work has been reviewed recently by Schroth et al. (2001). 7.3.1. Shade Legume trees have been used as shade plants for crops such as coffee (e.g., species of Inga in Central and South America) and cocoa (e.g., Albizzia lebbek (L.) Benth. in Ghana) for many years. A number of studies has shown that nitrogen fixation by Inga can be important for the associated crop (see references in Pennington and Fernandez, 1998). Moreover, recent work in Mexico (Romero-Alvarado et al., 2002) found that mixtures of shade trees, some legume and some not, were of equal benefit to the nutrient balance of the system when compared to mono-cropped legumes but, in addition, also enhanced biodiversity, including number of bird species present.

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7.3.2. Soil Properties Legume trees, e.g., Acacia senegal (L.) Willd in Senegal, can lead to improved soil nutrient content, a fact which is important in their use as fallow systems (Deans et al., 1999). In Costa Rica, growth of the timber tree, Terminalia amazonia (J.F.Gmel.) Exell (Combretaceae), planted on eroded, degraded pastures was considerably improved when interplanted with legume trees, especially Inga edulis Mart. (Nichols et al., 2001). One of the problems of soil cultivation for crops may be the formation of impermeable layers. As an alternative to mechanical breakage of such layers, Yunusa et al. (2002) looked at the effects of planting six native (to Australia) woody species, including three acacias. All, but the acacias in particular, had a marked effect in improving the hydraulic conductivity of the soil, allowing inter alia better root growth of subsequent crop species. On extremely sodic (pH 10.2) soils in northwestern India, a silvopastoral system, involving grasses and the legume trees, Acacia nilotica (L.) Del., Dalbergia sissoo Roxb. and Prosopis juliflora (Sweet) DC., showed the legume trees to lead to marked soil improvement (Kaur et al., 2002). Land reclamation, particularly following mining activity, is a priority in many areas. Australian acacias with dual mycorrhizas and nodules are proving to be particularly useful for this purpose. For example, when grown on residues from bauxite mines in Brazil, both A. holosericea G. Don and A. mangium in 22 months produced 4-to-5-times more above-ground biomass than several other species including the endemic A. angustissima (Mill.) Kuntze that only forms AM (Franco and Faria, 1997). At the opposite end of the scale, legumes, including acacias, may be used for biogeochemical prospecting. A recent study in a mountainous region of Pakistan found that a number of deep-rooted woody plants, including A. arabica Willd. (now usually known as A. nilotica subsp. indica (Benth.) Brenan), can take up Zn and Cu to varying amounts, which reflect the concentrations in the underlying rock, thus, enabling accurate targeting of mining activities (Naseem and Sheikh, 2002). In contrast, the ability of some tree legumes, especially dual mycorrhizal acacias, to grow on poor soils has led to severe problems in areas such as the Cape province of South Africa, where they have invaded the fragile Fynbos vegetation which is unique to the area (Stock et al., 1995; Holmes and Cowling, 1997). In other parts of South Africa, where these acacias (and other non-legume trees) have been grown for commercial timber, their fast growth is associated with high rates of water usage. This, in turn, is leading to major reductions in river flow in some catchment areas and so impeding economic growth (Le Maitre et al., 2002). The invasiveness of these acacias is enhanced by their ability to reproduce freely by both seed and vegetative methods. Prolific seeding has proved to be the downfall of Leucaena leucocephala in many countries, e.g., Cameroon (Kanmegne and Degrande, 2002), where it was introduced as an agroforestry plant and has turned out to be an invasive weed. Finally, the fact that nitrogen fixation can impact other reactions in the nitrogen cycle should not be ignored (see the chapters herein by van Spanning and Fiencke and by Bock). Nitrate run-off from agricultural soils following ploughing of pastures is well known, but a recent report from an experiments in Uganda, where nitrogen

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fixing and non-fixing trees were compared, found that the soils around the former produced up to four-times as much NO and N2O, both greenhouse gases, following rain (Dick et al., 2001). REFERENCES Allen, O. N., and Allen, E. K. (1981). The Leguminosae: A source book of characteristics, uses and nodulation. Madison, WI: University of Wisconsin Press, and London: Macmillan Publishers Ltd. Anon (1979). Tropical Legumes: Resources for the future. Washington, D.C.: National Academy of Sciences. Bala, A., and Giller, K. E. (2001). Symbiotic specificity of tropical tree rhizobia for host legumes. New Phytol., 149, 495-507. Barnet, Y. M., and Catt, P. C. (1991). Distribution and characteristics of root nodule bacteria isolated from Australian Acacia spp. Plant Soil, 135, 109-120. Barrios, E., and Herrera, R. (1994). Nitrogen cycling in a Venezuelan tropical seasonally flooded forest: soil nitrogen mineralization and nitrification. J. Trop. Ecol., 10, 399-116. Batzli, J. M., Graves, W. R., and van Berkum, P. (1992). Diversity among rhizobia effective with Robinia pseudoacacia L. Appl. Environ. Microbiol., 58, 2137-2143. Boddey, R. M., Peoples, M. B., Palmer, B., and Dart, P. J. (2000). Use of 15N natural abundance technique to quantify biological nitrogen fixation by woody perennials. Nutrient Cycling in Agroecosystems, 57, 235-270. Bonnier, C. (1960). Symbiose Rhizobium-légumineuses: Aspects particuliers aux régions tropicales. Ann. Inst. Pasteur, 98, 537-556. Burdon, J. J., Gibson, A. H., Searle, S. D., Woods, M. J., and Brockwell, J. (1999). Variation in the effectiveness of symbiotic associations between native rhizobia and temperate Australian Acacia: Within-species interactions. J. Appl. Ecol., 36, 398-408. Corby, H. D. L. (1988). Types of rhizobial nodule and their distribution among the Leguminosae. Kirkia, 13, 53-123. Deans, J. D., Digne, O., Lindley, D. K.. Dione, M., and Parkinson, J. A. (1999). Nutrient and organicmatter aacumulation in Acacia Senegal fallows over 18 years. For. Ecol. Manag., 124,153-167. Dick, J., Skiba, U., and Wilson, J. (2001). The effect of rainfall on NO and N 2O emissions from Ugandan agroforest soils. Phyton-Annales Rei Botanicae, 41, 73-80. DuPuy, D. (2001). Legumes of Madagascar. Kew, UK: Royal Botanic Gardens. Faria, S. M. de, and Lima, H. C. de (1998). Additional studies of the nodulation status of legume species in Brazil. Plant Soil, 200, 185-192. Faria, S. M. de, McInroy, S. G., and Sprent, J. I. (1987). The occurrence of infected cells, with persistent infection threads, in legume root nodules. Can. J. Bot., 65, 553-558. Founoune, H., Duponnois, R., and Bâ, R. (2002). Ectomycorrhization of Acacia mangium, Willd. and Acacia holosericea A. Cunn. ex G. Don in Senegal. Impact on plant growth, populations of indigenous microorganisms and plant parasitic nematodes. J. Arid Environ., 50, 325-332. Franco, A. A., and Faria, S. M. de. (1997). The contribution of N2-fixing tree legumes to land reclamation and sustainability in the tropics. Soil Biol. Biochem., 29, 897-903. Galiana, A., Balle, P., Kanga, A. N., and Domenach, A. M. (2002). Nitrogen fixation estimated by the N-15 natural abundance method in Acacia mangium Willd. inoculated with Bradyrhizobium sp and grown in silvicutural conditions. Soil Biol. Biochem., 34, 251-262. Giller, K. E. (2001). Nitrogen Fixation in Tropical Cropping Systems. 2nd Edition. Wallingford, UK: CAB International. Gross, E., Cordeiro, L., and Caetano, F. H. (2002). Nodule ultrastructure and initial growth of Anadenanthera peregrina (L.) Speg. var. falcate (Bneth.) Altschul plants infected with rhizobia. Ann. Bot., 90, 175-183. Högberg, P., and Piearce, G. D. (1986). Mycorrhizas in Zambian trees in relation to host taxonomy, vegetation type and successional patterns. J. Ecol., 74, 775-785. Holmes, P. M., and Cowling, R. M. (1997). The effects of invasion by Acacia saligna on the guild structure and regeneration capabilities of South African fynbos. J. Appl. Ecol., 34, 317-332.

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Kanmegne, J., and Degrande, A. (2002). From alley cropping to rotational fallow: Farmers involvement in the development of fallow management techniques in the humid forest zone of Cameroon. Agroforestry Systems, 54, 115-120. Kaur, B., Gupta, S. R., and Singh, G. (2002). Carbon storage and nitrogen cycling in silvopastoral ststems on a sodic soil in northwestern India. Agroforestry Systems, 54, 21-29. Lavin, M., Pennington, R. T., Klitgaard, B. B., Sprent, J. I., de Lima, H. C., and Gasson, P. E. (2001). The Dalbergioid legume (Fabaceae): Ddelimitation of a pantropical monophyletic clade. Amer. J. Bot., 88, 503-533. Le Maitre, D. C., Wilgen, B. W. van, Gelderblom, C. M., Bailey, C., Chapman, R. A., and Nel, J. A. (2002). Invasive alien trees and water resources in South Africa: Case studies of the costs and benefits of management. Forest Ecol. Management, 160, 143-159. Lorenzi, H. (1992). Árvores Brasileiras. SP Brasil: Editora Plantarum LTDA. Loureiro, M. F., Faria, S. M. de, James, E. K., Pott. A. A., and Franco, A. A. (1994). Nitrogen-fixing stem nodules of the legume, Discolobium pulchellum Benth. New Phytol., 128, 283-295. McInroy, S. G. (1997). Rhizobia for African species of Acacia: Characterisation, conservation and selection of inoculants. University of Dundee, UK: M.Sc. Thesis. Moreira, F. M. S, Haukka, K., and Young, J. P. W. (1998). Biodiversity of rhizobia isolated from a wide range of forest legumes in Brazil. Mol. Ecol., 7, 889-895. Naisbitt, T., James, E. K., and Sprent, J. I. (1992). The evolutionary significance of the genus Chamaecrista as determined by nodule structure. New Phytol., 122, 487-492. Naseem, S., and Sheikh, S. A. (2002). Biogeochemical prospecting of sulphide minerals in Winder Valley, Balochistan, Pakistan. Res. Geol., 52, 59-66. Nichols, J. D., Rosemeyer, M. E., Carpenter, F. L., and Kettler, J. (2001). Intercropping legume trees with native timber rapidly restores cover to eroded tropical pastures without fertilization. Forest Ecol. Management, 152, 195-209. Nick, G. (1998). Polyphasic taxonomy of rhizobia isolated from tropical tree legumes. University of Helsinki, Finland: Ph.D. Thesis. Odee, D. W., Njoroge, J., Machua, J., and Dart, P. (1998). Selective preference for nodulation and symbiotic nitrogen fixing potential of indigenous rhizobia with African and Australian acacias. In C. Elmerich, A. Kondorosi, and W. E. Newton (Eds.), Biological Nitrogen Fixation for the 21st Century, (pp. 673-674). Dordrecht, The Netherlands: Kluwer Acacemic Publishers. Odee, D. W., Haukka, H., McInroy, S. G., Sprent, J. I., Sutherland, J. M., and Young, J. P. W. (2002). Genetic and symbiotic characterization of rhizobia isolated from tree and herbaceous legumes grown in soild from ecologically diverse sites in Kenya. Soil Biol. Biochem., 34, 801-811. Oldfield, S., Lusty, C., and MacKinven, A. (1998). The World List of Threatened Trees. Cambridge, UK: World Conservation Press. Pennington, T. D., and Fernandez, E. C. M. (Eds.) (1998). The Genus Inga: Utilization. Kew, UK: Royal Botanic Gardens. Peoples, M. B., Faizah, A. W., Rerkasen, B. and Herridge, D. F. (Eds.) (1989). Methods for Evaluating Nitrogen Fixation by Nodulated Legumes in the Field. Canberra, Australia: ACIAR. Perreijn, K. (2002) Symbiotic Nitrogen Fixation by Leguminous trees in Tropical Rain Forest in Guyana. Ph.D. Thesis, University of Utrecht, The Netherlands: Tropenbos-Guyana Series 11. Pueppke, S. G., and Broughton, W. J. (1999). Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host-ranges. Mol. Plant-Microbe Interact., 12, 293-318. Ribeiro, J. E. L. da S., Hopkins, M. J. G., Vicenti, A., Sother, C. A., Costa, M. A. da S., Britto, J. M. de, et al. (1999). Flora da Reserva Ducke. Manaus, Brazil: INPA. Röhm, M., and Werner, D. (1992) Robinia pseudoacacia-Rhizobium symbiosis: Isolation and characterization of a fast nodulating and efficiently nitrogen fixing, Rhizobium strain. Nitrogen Fixing Tree Research Reports, 10, 193-197. Romero-Alvarado, Y., Soto-Pinto, L., García-Barrios, L., and Barrera-Gaytán, J. F. (2002). Coffee yields and soil nutrients under the shades of Inga sp. vs. multiple species in Chiapas, Mexico. Agroforestry Systems, 54, 215-224. Schroth, G., Lehmann, J., Rodrigues, M. R. L., Barros, E., and Macêdo, J. L. V. (2001). Plant-soil interactions in multistrata agroforestry in the humid tropics. Agroforestry Systems, 53, 85-102. Sen, D., and Weaver, R. W. (1984). A basis of different rates of N2-fixation by some strains of Rhizobium in peanut and cowpea root nodules. Plant Sci. Lett., 34, 239-246. Sprent, J. I. (1999). Nitrogen fixation and growth of non-crop species in diverse environments. Persp. Plant

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Ecol. Evol. System., 2, 149-162. Sprent, J. I. (2000). Nodulation as a taxonomic tool. In P. S. Herendeen and A. Bruneau (Eds.), Advances in Legume Systematics, Part 9 (pp. 21-44). Kew, UK: Royal Botanic Gardens. Sprent, J. I. (2001). Nodulation in Legumes. Kew, UK: Royal Botanic Gardens. Sprent, J. I., Geoghegan, I. E., Whitty, P. W., and James, E. K. (1996). Natural abundance of 15N and 13C in nodulated legumes and other plants in the Cerrado and neighbouring regions of Brazil. Oecologia, 105, 440-446. Sprent, J. I., and Parsons, R. (2000). Nitrogen fixation in legume and non-legume trees. Field Crops Res., 65, 183-196. Sprent, J. I., Sutherland, J. M., and Faria, S. M. de (1989). Structure and function of nodules from woody legumes. In C. H. Stirton and J. L. Zarucchi (Eds.) Monographs in Systematic Botany 29 (pp. 559578). St Louis, MO: Missouri Botanical Gardens. Ståhl, L., Nyberg, G., Högberg, P., and Buresh, R. J. (2002). Effects of planted tree fallow on soil nitrogen dymanics, above-ground and root biomass, N2-fixation and subsequent maize crop productivity in Kenya. Plant Soil, 243, 103-117. Stock, W. D., Wienand, K. T., and Baker, A. C. (1995). Impacts of invading N2-fixing Acacia species on patterns of nutrient cycling in two Cape ecosystems - Evidence from soil incubation studies and 15N natural abundance values. Oecologia, 101, 375-382. Subba Rao, N. S., and Yatazawa, M. (1984). Stem Nodules. In N. S. Subba Rao, N.S. (Ed.), Current Developments in Biological Nitrogen Fixation (pp. 101-110). New Delhi, India: Edward Arnold (see page 2, Yatazawa; Yatazawa is correct). Sutherland, J. M., Odee, D. W., Muluvi, G. M., McInroy, S. G., and Patel, A. (2000). Single and multistrain rhizobial inoculation of African acacias in nursery conditions. Soil Biol. Biochem., 32, 323-333. Turk, D., and Keyser, H. K. (1992). Rhizobia that nodulate tree legumes: Specificity of the host for nodulation and effectiveness. Can. J. Microbiol., 38, 451-460. Van Kessel, C. J. H. (1983). Effects of environmental and physiological factors in N2 fixation by Inga jinicuil and Trifolium species. University of Utrecht, The Netherlands. Ph.D. Thesis. Vega-Hernàndez, M., Pérez-Galdona, R., Dazzo, F. B., Jarabo-Lorenzo, A., Alfayate, M. C., and LeònBarrios, M. (2001). Novel infection process in the indeterminate root nodule symbiosis between Chamaecytysis proliferus (tagasaste) and Bradyrhizobium sp. New Phytol., 150, 707-721. Vitousek, P. M., Cassman, K., Cleveland, C., Crews, T., Field, C. B., Grimm, et al. (2002). Towards an ecological understanding of biological nitrogen fixation. Biogeochem., 57/58, 1-45. Yunusa, I. A. M., Mele, P. M., Rab, M. A., Schefe, C. R., and Beverly, C. R. (2002) Priming of soil structural and hydrological properties by native woody species, annual crops, and a permanent pasture. Austral. J. Soil Res., 40, 207-219. Zuzana, M., and Ward, D. (2002). Acacia trees as keystone species in Negev desert ecosystems. J. Veget. Sci., 13, 227-236.

Chapter 8

NITROGEN-FIXING TREES WITH ACTINORHIZA IN FORESTRY AND AGROFORESTRY R. O. RUSSO EARTH University, Costa Rica

1. INTRODUCTION Nitrogen-fixing trees with actinorhiza (or actinorhizal trees) form a group that is a key component in many natural ecosystems, agro-ecosystems, and agroforestry systems in the world, and that provides an important source of fixed nitrogen in these ecosystems. The general characteristics of the actinorhizal symbiosis, including aspects of nodule formation, co-evolution of both partners, and nitrogen fixation rates at field level of some selected species, as well as mycorrhizal associations in actinorhizal trees, will be described. In addition, topics related to actinorhizal trees in agroforestry, plus the role these woody perennial components may play in agroforestry systems, will also be explored, with emphasis on two genera, Casuarina and Alnus, widely known and used in this kind of production systems. The experience of the Central America Fuelwood Project, the case of Alnus acuminata in tropical highlands, and other uses of actinorhizal trees are also covered. Just as actinorhizal trees open new horizons for research and applications aimed toward sustainability, this chapter attempts to make a modest complementary contribution to the knowledge generated in the last four decades. Actinorhiza is the result of a symbiotic relationship between a special soil N2fixing actinomycete (filamentous bacteria of the order Actinomycetales of the genus Frankia) and fine plant roots; it is neither the actinomycete nor the root, but rather the structure or root nodule formed from these two partners. Because the result of the symbiotic association is a root nodule, the host plant is known as an actinomycete-nodulated plant or actinorhizal plant (Torrey and Tjepkema, 1979; Huss-Danell, 1997). Actinorhizal associations range from the arctic to the tropics and from the semi-desert to rainforest ecosystems. Actinorhizal plants can be found in forest, swamp, riparian, shrub, prairie, and desert ecosystems. In moving from warmer to colder climates, actinorhizal plants become more prevalent and seem to 143 D. Werner and W. E. Newton (eds.), Nitrogen Fixation in Agriculture, Forestry, Ecology, and the Environment, 143-171. © 2005 Springer. Printed in the Netherlands.

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fill the niche dominated by woody legumes in the tropics (Dawson, 1986). The actinorhizal trees are much less numerous than the vast group of the nitrogen-fixing legume trees. Even so, they are an important source of fixed nitrogen in their ecosystems and can fix from 2 to 300 kg N ha-1 yr-1. Despite of the size of the group, actinorhizal trees are important because: (a) their nitrogen-fixing ability is not restricted to one family but it expands to eight families; (b) most of the actinorhizal trees are good and sometimes aggressive colonizers that are capable of regenerating poor soils or disturbed sites, which are rapidly increasing in many tropical countries; and (c) tropical actiinorhizal trees, especially Casuarina and Alnus, produce not only timber but also firewood and charcoal, sometimes also providing shelter to the cattle, and are used for the protection and recovery of degraded soils (Diem et al., 1984). Many actinorhizal plants are pioneering species, like Alnus, which grows in moist environments, or Myrica, which grows on landslides, eroded slopes, and mined areas, or Casuarinas, which have been identified as nitrogen-fixing trees for adverse sites (NRC, 1984). This chapter will assess the realities and opportunities of actinorhizal trees in forestry and agroforestry, with an emphasis on tropical regions where the author has expertise and working experience. 2. GENERAL CHARACTERISTICS OF THE ACTINORHIZAL SYMBIOSIS Actinorhizal trees are defined by their ability to form N2-fixing root-nodule symbioses with the genus, Frankia. This association is typical for angiosperms but has not been reported in gymnosperms. Frankia is a Gram-positive, branched, septate, filamentous bacterium of the order Actinomycetales, which shows a complex pattern of differentiation in vitro and in the nodule (Berry, 1986). Frankia has also been defined as a diverse group of soil actinomycetes that have in common the formation of multi-locular sporangia, filamentous growth, and nitrogenasecontaining vesicles that are enveloped in multi-laminated lipid envelopes (Benson and Silvester, 1993). Frankia infects a host plant to form a nodule on the root system. In the symbiosis, Frankia provides the plant with a source of fixed nitrogen and, in return, the plant provides the Frankia with a carbon source. Although some 40 attempts had been made earlier (Baker and Torrey, 1979), the first confirmed Frankia strain was isolated in 1978 from nodules of Comptonia, using an enzymatic method (Callaham et al., 1978). Other methods, e.g., sucrose-density-gradient centrifugation (Baker et al., 1979), serial dilution (Lalonde, 1979), selective incubation (Quispel and Burggraaf, 1981), and osmium-tetroxide treatment (Lalonde et al., 1981), have successfully produced Frankia in pure culture. 2.1. Host Specificity Groups. Early on, compatibility groups of host plants and Frankia were thought to exist (Becking, 1974), however, later studies found that some isolates were capable of nodulating hosts from different “compatibility” groups (Lechevalier and Ruan, 1984) and this finding opened new horizons for research concerning host specificity

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(Berry, 1986). These newer studies often apply molecular-biology tools to questions related to the genetics, ecology, and evolution of actinorhizal symbiotic systems. Molecular phylogeny groupings of host plants were correlated with morphological and anatomical features of actinorhizal nodules in the past, but hostplant phylogenies that are based on molecular data soon revealed different relationships among host plants than have previously been assumed (Berry, 1994; Swensen and Mullin, 1997a ). Three host-specificity groups, Alnus, Elaeagnus, and Casuarina, have been identified and the majority of Frankia strains studied fell into one of these three cluster groups. The agreement between the phenotypic clusters and the genospecies described previously shows that the grouping may reflect the taxonomic structure of the genus, Frankia. Dobritsa (1998) validated this concept with a study in which thirty-nine selected Frankia strains, which belonged to different genomic species, were clustered on the basis of their in vitro susceptibility to 17 antibiotics, pigment production and ability to nodulate plants of the genus Alnus and/or the family Elaeagnaceae or the family Casuarinaceae. The author found differentiating phenotypic characters for some clusters that may be useful for species definition. Simultaneously, Huguet et al. (2000) carried out a study to identity Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis growing in close proximity and also from nodules of two additional stands with M. gale as the sole actinorhizal component. Using a genepolymorphism technique, they demonstrated that, at the first site, each host-tree species was nodulated by a different phylogenetic group of Francia. At the second site, they found that the M. gale-strains belonged to yet another group and were relatively low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. It should be remembered here that plants in the Myricaceae family are considered to be promiscuous hosts because several species are effectively nodulated by most isolated strains of Frankia in the greenhouse. This observation has led to the hypothesis that plants in the Myricaceae family have sufficient diversity to serve as a reservoir host for Frankia strains that infect plants from other actinorhizal families (Clawson and Benson, 1999). These issues have contributed to the development of new hypotheses on the origin and evolution of actinorhizal symbiotic systems and have initiated the notion that nitrogen-fixing endosymbionts of actinorhizal plants can interact with a very broad range of unrelated host-plant genotypes (Kohls et al., 1994). Furthermore, a model for actinorhizal specificity has been proposed that includes different degrees of specificity of host-symbiont interactions, ranging from fully compatible to incompatible, and also that actinorhizal plants undergo feedback regulation of symbiosis, involving at least two different consecutive signals that control root nodulation (Wall, 2000). Further, the use of heterologous probes in combination with nucleotide-sequence analysis have allowed a number of nif genes to be mapped on the Frankia chromosome and this work will ultimately contribute to our understanding of nif-gene regulation in Frankia (Mullin and Dobritsa, 1996). Actinorhizal nodules are ontogenically related to lateral roots and may result from modification of the developmental pathway that leads to lateral-root formation

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(Goetting-Minesky and Mullin, 1994). This type of root nodule is characterized by differentially expressed genes, which supports the idea of the distinctiveness of this new entity, called "Actinorhiza", a name that refers to both the filamentous bacteria and to the root location of nitrogen-fixing nodules (Wall, 2000).

Figure 1. Root Nodules on Alnus glutinosa. (Photo: R.O. Russo)

2.2. Infection Pathways. Two pathways for root infection have been described for compatible Frankia interactions; they are either root-hair infection or intercellular penetration. The functional physiology of nodule morphogenesis has been reviewed by Berry (1994). As stated by this autor, nodules are initiated in actinorhizal plants either via root infection by Frankia or by intercellular invasion (Miller and Baker, 1985) and that the mode of infection is host-determined because single strains of Frankia have been shown to nodulate different hosts by different infection mechanisms (Miller and Baker, 1986). Premature stages of the developing infection embrace Esther deformations of the root-hair wall or, in the case of intracellular penetration, the

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release of host extracellular pectic polysaccharides (Liu and Berry, 1991). Host cortical-cell proliferation is followed by expansion and even hypertrophy of groups of cells infected by Frankia. The endosymbiont penetrates cortical cells after meristematic growth, probably during cell expansion. Within the host cells, Frankia proliferates and vesicles are formed, wherein nitrogen fixation takes place (except with the Casuarinaceae family). Frankia strains can be classified in two groups, those that form spores (sp+) within the nodules (spore-positive nodules) and those that do not (sp-) and so produce spore-negative nodules (Schwintzer, 1990). It appears that sp+ strains are less common than sp-. The morphology of the vesicles is relatively constant in culture but, in symbiotic conditions when interacting with the host plant, it is modified to give a diverse array of vesicles shapes (Benson and Silvester, 1993). Another line of research involves the function of flavonoid compounds on the patterns of nodulation by Frankia, e.g., Benoit and Berry (1997) found that flavonoid-like compounds from seeds of red alder (Alnus rubra) influenced host nodulation. Independent of the infection mechanism, some authors support the concept that the opening (or closing) of the window of susceptibility for infection and nodule development in the growing root are signal-mediated processes (Wall, 2000), even though rhizosphere nodulation signals have not been as well characterized for actinorhizal plants as they have been for legumes. Recently, the steps in nodule formation for actinorhizal and rhizobial symbioses have been compared and phylogenetic aspects of nodulating both actinorhizal and legume plants described (Gualtieri and Bisseling, 2000). 2.3. External Factors That Impact Nodulation. Many factors intervene in the process of nodulatin and nodule formation. As for legumes, fixed-nitrogen levels in the substrate inhibit both nodulation and N2 fixation in actinorhizal plants (Huss-Danell, 1997). This effect had been observed in 1975 by Bond and Mackintosh, who reported that nitrate inhibited both nodule biomass and nitrogenase activity in Hippophaë rhamnoides and Coriaria arborea (Gentili and Huss-Danell, 2002). The same pattern was also observed by Arnone et al. (1994), who used a split-root system that allowed them to distinguish between local and systemic effects of fixed-N compounds. They found that nodulation was inhibited locally by nitrate in Casuarina cunninghamiana. But fixed-N compounds are not the only impacting factor. Phosphorus compounds interact with fixed-N compounds and together affect nodulation. This interaction effect between ammonium nitrate and phosphate on nodulation by the intercellular infection was studied by Gentili and Huss-Danell (2002). They found that phosphate modifies the effects of fixed-nitrogen compounds on nodulation in split-root systems of Hippophaë rhamnoides. Further, the inhibition of nodulation by fixed-N compounds was systemic for both nodule number and nodule biomass and the high phosphate level had a systemic stimulation on nodule number and biomass. In this study, phosphate prevented the systemic inhibitoin by fixed-N compounds. The stimulation by phosphate was specific to nodulation and not

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simply mediated via plant growth. This study, however, did not allow the authors to make conclusions on the effect of both fixed-N and P compounds on N2 fixation. In addition to root nodulation, aerial nodulation has been observed in Casuarina by a number of authors. Prin et al. (1991a; 1991b) reported aerial nodules on the trunks of Casuarina cunnimghamiana. Then, Valdés and Cruz Cisneros (1996) described stem nodulation of Casuarina in Mexico and Bertalot and Mendoza (1998) observed aerial nodulation in Casuarina equisetifolia that was shading pastures in the inlands of the State of Sao Paulo in Brazil. The authors described them as stem protuberances, which contained actinorhizal nodules and adventitious roots that were 3-8 cm thick. They covered irregularly up to 20 percent of the trunk surface below breast height and tested positively by the acetylene-reduction assay. 2.4. Co-Evolution of the Symbionts. Taking into consideration that two genomes, one from the host and one from the actinomycete, are involved in the host-endophyte interactions, this is an intricate symbiotic relationship. Many factors are involved in the actinorhizal symbiosis; these include host-plant physiology, host compatibility, host specificity in terms of both infectivity or affectivity, ability to promote host-plant growth, and interactions of the microsymbiont with other microorganisms inside or outside the host root. All these interactions have led to a coevolution process between the two genomes. Considerable evidence exists to support the hypothesis that coevolution has taken place through mutual interaction of host plants and Frankia populations in the soil to produce the most efficient symbiotic associations (Lie et al., 1984). Several research groups interested in coevolution have studied Frankia, when associated with host plants of different genera and families, and have found some correspondence between the bacterial and plant phylogenies, which suggests coevolution (Benson et al., 1996). Although an exploratory approach, based on genome interactions, is helpful in looking at this problem, the situation is much more complicated than it appears, mainly because the host-plant genome is more complex than that of the prokaryote. Studies on co-evolution between Frankia populations and host plants in the family Casuarinaceae were carried out by Simonet et al., (1999), who deduced patterns of global dispersal. The ecological significance of considering the evolution of both symbiotic partners is that the complete symbiotic unit can explore a wider environmental range. This is the situation for those actinorhizal trees that grow in: (i) poor, disturbed, or degraded soils; (ii) in nitrogen-poor sites, such as sandy soils, disturbed soils, and wet soils; or (iii) as pioneer vegetation at early stages of plant succession following disturbances, such as eruptions, flooding, landslides, and fires. 3. HOST BOTANICAL FAMILIES Actinorhizal hosts described to date are trees and shrubs that, according to the classification scheme of Cronquist (1981), which is based primarily on the analysis

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of morphological characters, are distributed among four subclasses, Rosidae, Hamamelidae, Magnoliidae and Dilleniidae, in eight families of angiosperms that include 25 genera and over 200 plant species. This broad phylogenetic distribution of nodulated actinorhizal plants has created the notion that nitrogen-fixing endosymbionts, particularly those of actinorhizal plants, can interact with a very broad range of unrelated host-plant genotypes (Swensen and Mullin, 1997b). Several reviews on the biology and development of these actinorhizal associations have been published in recent years (Benson and Silvester, 1993; Berry, 1994; Schwintzer and Tjepkema, 1990). Not all genera in these families and not all species in the 25 genera fix atmospheric N2. In certain environments, species of these genera may become more abundant and ecologically significant. According to Benson and Clawson (2000), nodulating families occur sporadically within the families; in the Casuarinaceae, Coriariaceae, Datiscaceae, Elaeagnaceae, and Myricaceae, all or most genera are nodulated, whereas nodulation occurs only occasionally in the Betulaceae, Rhamnaceae, and Rosaceae. Table 1 summarizes the taxonomy of the actinorhizal genera with their distribution. 4. NITROGEN FIXATION IN ACTINORHIZAL TREES Measurement of N2 fixation in actinorhizal trees (Table 2) is still a difficult task because no simple and accurate methods to determine annual rates are available. Conventionally, N2 fixation has been measured by the acetylene-reduction assay, which is based on measuring the production of ethylene from acetylene, catalyzed by nitrogenase, with a gas chromatograph. The enzyme, within the N2-fixing organisms, can also reduce a number of other compounds besides N2 and acetylene. However, several other methods have been used, including N accretion, N difference, 15N isotope dilution, and 15N natural abundance. Each of the methods has its own assumptions and inaccuracies (Table 3). Of these, the 15N stable-isotope methods are currently considered to have the least drawbacks for the quantitative measurement of biological nitrogen fixation (BNF) (Warembourg, 1993). Their advantages are based on: (i) the capacity to measure BNF cumulatively over more than one growing season; (ii) the high degree of precision of stable isotopes; and (iii) the capability of assessing the relative efficiency of N2 fixation, i.e., the proportion of plant-N derived from fixation (Ndff). Stable-isotope methods are not without their limitations, however. Several assumptions, which primarily involve the selection of the reference plant, must be satisfied to ensure an accurate measurement of BNF. Reference plants are required in order to estimate the relative proportion of plant-N derived from fixation versus uptake of soil-N. They must have a similar rooting profile, a similar timing of Nuptake, and a similar internal isotopic discrimination to those of the N2-fixing plants. Finding an appropriate reference plant is always difficult. In the case of actinorhizal plants, the Ndff determined by 15N isotope-dilution methods averaged 87% for Ceanothus velutinus and 83% for Purshia tridentata (Busse, 2000a; 2000b). Other studies have reported values of 68 to 100 percent for Alnus glutinosa

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for N derived from fixation (Beaupied et al., 1990; Domenach et al., 1989) and 4867% for Casuarina equisetifolia (Gauthier et al., 1985, Parrotta et al., 1994). Table 1. Taxonomy of the Actinorhizal Genera with theirDistribution.

Subclass* Hamamelidae

Family Betulaceae

Genus Alnus

Casuarinaceae

Allocasuarina Casuarina

Ceuthostoma Gymnostona

Myricaceae

Rosidae

Elaeagnaceae

Rhamnaceae

Comptonia Myrica Elaeagnus Hippophae Shepperdia Adolphia Ceanothus Colletia

Discaria

Rosaceae

Kenthrothamnus Retanilla Talguena Trevoa Cercocarpus Chamaebatia Cowania Dryas

Magnolidae

Coriariaceae

Purshia Rubus Coriaria

Dillenidae

Datiscaceae

Datisca

Present distribution Europe, N. America, Highlands of Central and South America, scattered in North Africa. Australia, primarily S and SW Australia, troprical Asia, Pacific Islands. Sumatra to New Caledonia, Fiji and Cape York Pen., Australia. Europe, Asia, North America. Europe, Asia, North America. North America. Widespread tropical, subtropical, temperate. Southern South America. North America. Temperate and tropical South America. South America, New Zealand, Australia. Argentina, Bolivia. Southern Soth America. Chile. Andes of South America. West and S-W USA, Mexico. Sierra Nevada of West USA. USA, México Artic mountains, North Temperate Zone North America. North Temperate Zone Mediterranean, Japan, China, New Zealand, Chile, Mexico. Mediterranean to Himalayas and Central Asia, S-W USA.

Classification according to Cronquist (1981), Brewbaker et al. (1990), and Benson and Clawson (2000).

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Table 2. Rates of Symbiotic N2 Fixation of Some Actinorhizal Trees and Shrubs at Field Level

Actinorhizal species Alnus acuminata (A. jorullensis)

kg N fixed ha–1 yr–1

References

279

Carlson and Dawson, 1985

Alnus glutinosa

40 – 53

Cote and Camire 1984; Hansen and Dawson 1982

Alnus rubra

85 – 320

Casuarina equisetifolia

12 – 110

Coriaria arborea

192

Dommergues, 1990

Ceanothus velutinus

4 -100

McNabb and Cromack 1983; Youngberg and Wollum 1976; Zavitkovski and Newton 1968; Busse 2000a; 2000b 15N

Purshia tridentata

1

Busse 2000a; 2000b 15N

Cole et al. 1978; Newton et al. 1968 Dommergues 1987 ; Gauthier et al. 1985 ; Diem and Dommergues 1990 ; Baker 1990a

Table 3. Comparison of Methods of Estimating Nitrogen Fixation. After I. Watanabe (2000) lecture in Cantho University, Vietnam. Retrieved June 6, 2002, from http://www.asahi-net.or.jp/~it6i-wtnb/BNF.html#ch4

Method

Advantages

Total N balance

Simplest

15

N2 incorporation Acetylene reduction 15

N dilution

Natural abundance

Substrate addition

Most direct Simple, highly sensitive Throughout growing season Simple, no disturbance to system Difference in 15N content is large

Disadvantages Low sensitivity including other inputs. Expensive, only for short period Indirect, semiquantitative Only N Fixation in plant Varies with reference plants

Sensitivity Lowest

Highmoderate High High-low

Only slight difference in 15 N content

Low

Change of 15N in time and space in soil

Moderate

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The rates of nitrogen fixation measurable by the acetylene-reduction assay (ARA) in nodules of actinorhizal genera, such as Alnus and Eleagnus, range between 10-90 ȝmol ethylene g–1 nodule fresh weight h–1 (Berry, 1994). Other values of ARA reported for 120-days-old Alnus acuminata seedlings, which were inoculated with a crushed nodule suspension, were between 32.5 and 86.4 ȝmol ethylene g–1 nodule fresh weight h–1 (Russo and Berlyn, 1989; see Figure 2) and are consistent with the above mentioned range. However, variability is very high and accuracy has been challenged because acetylene, in some cases, inhibits nitrogenase activity. In other cases, the ARA technique is used inappropriately. Furthermore, conversion from acetylene-reduction to N2-fixation values often uses the routinely assumed conversion ratio of 3:1-4:1 (due to H2 production under N2), however, conversion rates can fluctuate depending on a number of factors and are not constant. Finally, to extrapolate laboratory or greenhouse values to field conditions in terms of kg N ha–1 yr –1 can be a significant source of error (Giller, 2001).

Figure 2. In Alnus acuminata seedlings inoculated with Frankia, nodules appear two weeks after inoculation and thereafter the rate of growth and interval of appearance of leaves is modified. Nodulated seedlings (inoculated) at 50 days have higher total height (P